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United States Patent |
5,281,522
|
Senyei
,   et al.
|
January 25, 1994
|
Reagents and kits for determination of fetal fibronectin in a vaginal
sample
Abstract
This invention relates to methods, reagents and kits for detection of
normal or ectopic pregnancy, the termination of pregnancy, or increased
risk of preterm labor and rupture of membranes. Each embodiment involves
sampling from the vaginal cavity, and determining the presence or absence
of a specific analyte in the test sample. Sandwich or competition assay
procedures can be used. Reagents and reagent kits for the above assays are
included. The kit contains anti-(fetal fibronectin) antibody and an
anti-fibronectin antibody.
Inventors:
|
Senyei; Andrew E. (San Juan Capistrano, CA);
Teng; Nelson N. H. (Hillsborough, CA)
|
Assignee:
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Adeza Biomedical Corporation (Sunnyvale, CA)
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Appl. No.:
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628282 |
Filed:
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December 14, 1990 |
Current U.S. Class: |
435/7.9; 436/518; 436/528; 436/536; 436/543; 436/547 |
Intern'l Class: |
C12Q 001/00; G01N 033/53; G01N 033/543 |
Field of Search: |
436/547,536,518,528,543
435/7.9
|
References Cited
U.S. Patent Documents
4313734 | Feb., 1982 | Leuvering.
| |
4347311 | Aug., 1982 | Schmitz | 435/810.
|
4353982 | Oct., 1982 | Gomez et al. | 435/810.
|
4486530 | Dec., 1984 | David et al.
| |
4632901 | Dec., 1986 | Valkirs et al.
| |
4675286 | Jun., 1987 | Calenoff.
| |
4894326 | Jan., 1990 | Matsuura et al. | 435/7.
|
Other References
Boehringer Mannheim, "Antibodies and Reagents for Immunochemistry,"
Biochemica, pp. 129-134 (1989).
Wisdom, G.,. "Enzyme-Immunoassay," Clin. Chem., pp. 1243-1255 (1976).
Gahl, et al, Obstet. Gynecol., 60:297-304 (1982).
Grudzinskas, et al (ed.), Pregnancy Proteins, New York: Academic Press
(1982).
Hess, et al, Obstet. Gynecol., 68:25-28 (1986).
Huber, et al, British J. Obstet. Gynecol., 90:1183-1185 (1983).
Konickx, et al, British J. Obstet. Gynecol., 88:607-610 (1981).
Kuusela, et al, Scand. J. Immunol., 12:331-337 (1980).
Matsuura, et al, Proc. Natl Acad. Sci. USA, 82:6517-6521 (1985).
Nakabayashi, et al, Cancer Res., 42:3858-3863 (1982).
Oellerich, M., J. Clin Chem. Clin. Biochem., 22:895-904 (1985).
Rochelson, et al, Obstet. Gynecol., 69:163-165 (1987).
Ruoslahti, et al, Int. J. Cancer, 27:763-767 (1981).
Wisdom, G., Clin. Chem., 22:1243-1255 (1976).
|
Primary Examiner: Rosen; Sam
Attorney, Agent or Firm: Skjerven, Morrill, MacPherson, Franklin & Friel
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation In Part of U.S. application Ser. No.
07/274,268, now abandoned, filed Nov. 18, 1988; U.S. application Ser. No.
07/244,969, now U.S. Pat. No. 5,096,830 filed Sep. 15, 1988; U.S.
application Ser. No 7/274,267 filed Nov. 18, 1988 now U.S. Pat. No.
5,223,440; and U.S. application Ser. No. 07/282,426 filed Dec. 12, 1988
now U.S. Pat. No. 5,185,270. The inventors of the above-identified
applications are ANDREW E. SENYEI and NELSON N. H. TENG. Each of those
applications is incorporated herein by reference in its entirety.
Claims
I claim:
1. A kit for detecting fetal fibronectin in a vaginal sample comprising:
a. an anti-(fetal fibronectin) antibody;
b. an anti-fibronectin antibody,
wherein either said anti-(fetal fibronectin) antibody or said
anti-fibronectin antibody is adhered to an insoluble support; and
c. a device selected from the group consisting of a vaginal sample
collection device, a vaginal sample filtration device, and a vessel
containing a vaginal sample diluent.
2. The kit of claim 1 wherein said anti-(fetal fibronectin) antibody is a
monoclonal antibody.
3. The kit of claim 2 wherein said anti-(fetal fibronectin) antibody is
FDC-6.
4. The kit of claim 1 wherein said anti-fibronectin antibody is a
polyclonal antibody.
5. The kit of claim 4 wherein said anti-fibronectin antibody is labeled.
6. The kit of claim 5 wherein said label is an enzyme.
7. The kit of claim 6 wherein said enzyme is alkaline phosphatase.
8. The kit of claim 7 wherein said kit additionally comprises enzyme
substrate.
9. The kit of claim 8 wherein said enzyme substrate is phenolphthalein
monophosphate.
10. The kit of claim 5 wherein said label is colloidal gold.
11. The kit of claim 1 wherein said insoluble support comprises a
microtiter plate or a strip of wells of a microtiter plate.
12. The kit of claim 11 wherein said kit additionally comprises a
microtiter plate cover.
13. The kit of claim 11 wherein said kit includes a strip of wells of a
microtiter plate and additionally comprises a holder for said strip.
14. The kit of claim 1 wherein said kit additionally comprises a positive,
control.
15. The kit of claim 14 wherein said positive control is amniotic fluid of
a known fetal fibronectin concentration.
16. The kit of claim 15 wherein said fetal fibronectin concentration is
from about 10 to about 100 ng/mL.
17. The kit of claim 16 wherein said amniotic fluid is diluted in 0.05M
Tris buffer pH 7.4, 1% bovine serum albumin, 0.15M sodium chloride, 0.02%
Sodium Azide, 5 mM ethylenediamine tetraacetic acid, 1 mM
phenylmethylsulfonyl fluoride, and 500 Kallikrein Units/ml of Aprotinin.
18. The kit of claim 1 wherein said kit additionally comprises a negative
control.
19. The kit of claim 18 wherein said negative control is 0.05M Tris buffer
pH 7.4, 1% bovine serum albumin, 0.15M sodium chloride, 0.02% Sodium
Azide, 5 mM ethylenediamine tetraacetic acid, 1 mM phenylmethylsulfonyl
fluoride, and 500 Kallikrein Units/ml of Aprotinin.
20. The kit of claim 1 wherein said kit additionally comprises at least one
sample filtering device.
21. The kit of claim 20 wherein said sample filtering device dispenses a
predetermined volume of filtered sample.
22. The kit of claim 1 wherein said kit additionally comprises a rinse
buffer.
23. The kit of claim 22 wherein said rinse buffer is 0.02M Tris, 0.08M
sodium chloride and 0.05% Tween-20.
24. The kit of claim 23 wherein said rinse buffer additionally comprises
sodium azide.
25. The kit of claim 23 wherein said rinse solution is packaged in
concentrated form.
26. The kit of claim 1 wherein said unsoluble support is a membrane.
27. The kit of claim 26 wherein said membrane is nylon.
28. The kit of claim 26 wherein said membrane is placed over an absorbent
layer.
29. The kit of claim 28 wherein a flow control layer is intermediate said
membrane and said absorbent layer.
30. The kit of claim 26 wherein said kit additionally comprises a sample
filtering device which contains labeled anti-fibronectin antibody.
31. The kit of claim 7 wherein the alkaline phosphatase-labeled
anti-fibronectin antibody is in an enzyme conjugate buffer comprising 50
mM Tris, pH 7.2; 2% D-Sorbitol; 2% bovine serum albumin; 0.01% Tween-20; 1
mM MgCl.sub.2 ; 0.1% ZnCl.sub.2 ; and optionally 0.1% sodium azide.
32. The kit of claim 9 wherein the phenolphthalein monophosphate is in an
enzyme substrate solution containing 2-amino-2-methyl-1-propanol and
MgCl.sub.2.
33. The kit of claim 32 wherein said enzyme substrate solution comprises 1
mg/ml phenolphthalein monophosphate; 0.4M 2-amino-2-methyl-1-propanol, pH
10; 0.1 mM MgCl.sub.2 ; and, optionally 0.2% sodium azide.
34. The kit of claim 10 wherein the gold-labeled anti-fibronectin antibody
conjugate is in a protein matrix in a conjugate buffer comprising 15 mM
Tris; 0.1% Tween 20; 0.2% polyethylene glycol; 8% polyvinylpyrrolidine;
and, optionally 0.04% thimerosal.
35. The kit of claim 34 wherein the protein matrix comprises 2% bovine
serum albumin.
36. The kit of claim 35 wherein said antibody conjugate is lyophilized.
37. The kit of claim 36 wherein said kit includes a conjugate
reconstitution buffer.
38. The kit of claim 37 wherein said conjugate reconstitution buffer
comprises 100 mM sodium acetate.
39. The kit of claim 1 wherein said device is a vaginal sample collection
device.
40. The kit of claim 1 wherein said device comprises a swab.
41. The kit of claim 40 wherein said swab is a dacron swab.
42. A kit for detecting a fetal fibronectin in a vaginal sample comprising:
a. an anti-(fetal fibronectin) antibody adhered to a well of a microtiter
plate;
b. an alkaline phosphatase-labeled anti-fibronectin antibody in an enzyme
conjugate buffer comprising 50 mM Tris, pH 7.2; 2% D-Sorbitol; 2% bovine
serum albumin; 0.01% Tween-20; 1 mM MgCl.sub.2 ; 0.1% ZnCl.sub.2 ; and
optionally 0.1% sodium azide;
c. phenolphthalein monophosphate is in a enzyme substrate solution
comprising 1 mg/ml phenolphthalein monophosphate; 0.4M
2-amino-2-methyl-1-propanol, pH 10; 0.1 mM MgCl.sub.2 ; and, optionally
0.2% sodium azide; and
d. a device selected from the group consisting of a vaginal sample
collection device, a vaginal sample filtration device, and a vessel
containing a vaginal sample diluent.
43. The kit of claim 42 wherein the kit additionally contains a rinse
buffer concentrate comprising 0.1M Tris, pH 7.4; 4.0M NaCl; 2.5% Tween-20;
and, optionally 1.0% sodium azide.
44. The kit of claim 42 wherein the kit additionally comprises a positive
and a negative control.
45. A kit for detecting a fetal fibronectin in a test sample comprising:
a. an anti-(fetal fibronectin) antibody adhered to a nylon membrane, said
membrane placed over an absorbent layer and having a flow control layer
intermediate said membrane and said absorbent layer;
b. a lyophilized gold-labeled anti-fibronectin antibody conjugate
comprising 15 mM Tris; 2% bovine serum albumin; 0.1% Tween 20; 0.2%
polyethylene glycol; 8% polyvinylpyrrolidine; and, optionally 0.04%
thimerosal; and
c. a reconstitution buffer comprising 100 mM sodium acetate.
46. The kit of claim 45 wherein said membrane additionally includes a
control region comprising membrane-affixed fibronectin.
Description
FIELD OF THE INVENTION
This invention relates to reagents and kits for immunological detection of
normal and ectopic pregnancy; the termination of pregnancy; and increased
risk of preterm labor and rupture of membranes.
BACKGROUND OF THE INVENTION
A wide variety of tests have been developed for the determination of
pregnancy. Commercial early pregnancy determinations generally involve
assay of urine or serum. Home pregnancy tests for hCG (human chorionic
gonadotropin) in urine include a variety of enzyme immunoassays,
hemagglutination inhibition, and antibody-indicator agglutination tests
which are effective to indicate pregnancy from 0 to 7 days after a missed
period. Confirmation by physician is recommended, particularly to
determine abnormal gestation such as ectopic pregnancy.
HCG is produced by the fetal trophoblast and passes from the fetal blood
into the mother's blood through the intervillous space in the placenta.
HCG levels in maternal blood and urine are often detectable at about 3
weeks. The sensitivity of serum or urine hCG tests is limited because the
amount of hCG produced is determined by the amount of trophoblastic
tissue, and by dilution of the hCG in the maternal fluids. Until
development of the .beta.-hCG specifically binding antibody,
cross-reaction with LH (luteinizing hormone) also placed a limit on the
level of sensitivity.
We have discovered that normal uterine pregnancies can be reliably
determined early in the gestation cycle by testing a sample removed from
the vicinity of the cervical canal, cervical os or posterior fornix of the
vagina, preferably the external cervical os or posterior fornix, for the
presence of fetal restricted antigens, that is, compounds or materials
which are produced in the placental tissue and which do not pass in any
substantial amounts into the maternal blood. Included in this class of
antigens are fetal fibronectins.
We have discovered that ectopic pregnancy can be determined by testing a
sample removed from the vicinity of the cervical canal or cervical os for
the presence of fetal restricted antigens, that is, compounds or materials
which are produced in the placental tissue and which do not pass in any
substantial amounts into the maternal blood. Included in this class of
materials are fetal fibronectins. If the fetal restricted antigens are
substantially depressed in a sample from a person who is tested positive
for pregnancy by a blood or urine test for pregnancy, an ectopic pregnancy
is indicated.
Determination of the presence of ex vivo products of conception in uterine
tissue removed in therapeutic or spontaneous abortion is critically
important to confirm the existence of uterine pregnancy and the
termination thereof, and to rule out the presence of an ectopic pregnancy.
If levels of fetal associated antigens in maternal serum or urine indicate
pregnancy, and the uterine tissue removed during a therapeutic abortion
does not contain products of conception, a possible ectopic pregnancy is
indicated. Presence of products of conception in uterine discharge
associated with indicator of spontaneous abortion confirms the abortion,
while the absence thereof indicates a continuation of pregnancy. Usual
immunoassay techniques for determining the presence of fetal associated
antigens in test samples derived from the vaginal cavity are not reliable
for indicating the presence of products of conception since these samples
typically contain maternal blood. Pregnancy antigens and fetal antigens
are usually present in maternal blood as well as in fetal and placental
tissue.
Determination of impending preterm births is critical for increasing
neonatal survival of preterm infants. Detection of rupture of the amniotic
membrane is important in distinguishing true and false labor. When the
rupture is small and the volume of amniotic liquid escaping is small, the
rupture is often undetected. Accepted methods for detecting ruptured
membranes are subjective, not sufficiently sensitive, and not specific. An
embodiment of this invention for detection of increased risk of preterm
labor and rupture of the amniotic membrane after week 20 of pregnancy is
directed to an assay of a test sample removed from the vicinity of the
posterior fornix, cervical canal, or cervical os.
SUMMARY OF THE INVENTION
The method of this invention is used to determine the presence and/or
status of a pregnancy. The method herein is used to determine the presence
of a diagnostic indicator in a sample derived from the vaginal cavity, and
comprises:
(a) a method for determining normal uterine pregnancy during the first 20
weeks of pregnancy comprising obtaining a test sample in the vicinity of
the cervical canal or cervical os, and determining the presence of a fetal
restricted antigen in the sample;
(b) a method for determining fallopian pregnancy comprising obtaining a
test sample in the vicinity of the cervical canal or cervical os from a
pregnant patient during the first 20 weeks of pregnancy, and determining
the absence of a fetal restricted antigen in the sample;
(c) a method for determining ex vivo products of conception comprising
obtaining a test sample expelled or removed from the uterus, and
determining the presence of a fetal restricted antigen in the sample; or
(d) a method for determining increased risk of preterm labor or fetal
membrane rupture comprising obtaining a test sample in the vicinity of the
posterior fornix, cervical canal or cervical os, from a patient after week
20 of pregnancy, and determining the presence of a fetal restricted
antigen in the sample.
Reagents for use with the assays of the invention include labeled and
unlabeled anti-(analyte) antibodies, i.e., anti-(fetal restricted antigen)
antibodies such as anti-(fetal fibronectin) antibodies; anti-(analyte
class) antibodies, i.e., anti-(fetal restricted antigen class) antibodies
such as anti-(fibronectin) antibodies, and the like. Other reagents for
use with the assays of the invention include insoluble supports to which
are adhered the anti-(analyte) antibodies, i.e., anti-(fetal restricted
antigen) antibodies such as anti-(fetal fibronectin) antibodies;
anti-(analyte class) antibodies, i.e., anti-(fetal restricted antigen
class) antibodies such as anti-(fibronectin) antibodies, and the like.
Labeled or unlabeled reagent fetal restricted antigens are also reagents
of this invention. Reagents for use with the assays of this invention also
include insoluble supports to which are adhered reagent analyte, i.e.,
insoluble supports to which are adhered fetal restricted antigens.
Reagents for use with the assays also include immunoassay reagents such as
labeled secondary antibody, positive and negative controls, label
development reagents such as enzyme substrate, and wash buffer.
This invention includes kits comprising one of the above reagents, alone,
in combination with another reagent or in combination with supplies such
as sample preparation devices. The reagents can be present in any suitable
form in the kit, for example in containers, packages, and the like.
DETAILED DESCRIPTION OF THE INVENTION
The method of this invention for determining the presence and/or status of
a pregnancy comprises determining the presence of a fetal restricted
antigen, in a test sample removed from the vaginal cavity in the vicinity
of the posterior fornix, cervical canal, or cervical os, and especially at
the cervical canal or cervical os. Specific embodiments of this invention
are used to determine normal uterine pregnancy, ectopic pregnancy, the
occurrence of a therapeutic or spontaneous abortion, and increased risk of
preterm labor or rupture of the membranes.
Reagents and kits useful in performing the method are also described.
FETAL RESTRICTED ANTIGEN TEST METHODS AND REAGENTS
A fetal restricted antigen testing method involves the detection of a fetal
restricted antigen, that is, a uniquely fetal or placental focused
material, in a test sample removed in the vicinity of the posterior
fornix, cervical canal or cervical os. We have discovered that detectable
amounts of these materials are present in such a sample. Since the fetal
restricted antigens are not present in significant quantities in the
maternal blood, the presence of maternal blood in the sample does not
interfere with the test.
The term "fetal restricted antigen" as used herein is defined to mean a
uniquely fetal or placental derived material, which is either not present
in maternal serum, plasma or urine, or is not present in significant
amounts in maternal serum, plasma or urine. Any substance meeting this
definition is intended to be included within the meaning of the term,
including both immunogenic materials and proteins and other substances
which are not immunogenic in their purified form but which have unique
epitopes which can be selectively bound with antibodies specific or
selective thereto. An example of a fetal restricted antigen is the fetal
fibronectin which binds specifically with the FDC-6 monoclonal antibody
described by H. Matsuura and S. Hakomori, Proc. Natl. Acad. Sci. USA
82:6517-6521 (1985). Production of the hybridoma (deposited at the
American Type Culture Collection as accession number ATCC HB 9018) which
produces FDC-6 antibody is also described in detail in U.S. Pat. No.
4,894,326 issued Jan. 16, 1990 to Matsuura et al.
The term "fetal restricted antigen class" as used herein is defined to mean
a class or group of antigens of which the fetal restricted antigen is a
member. For example, fetal fibronectin is a fetal restricted member of the
human fibronectin group or class.
The term "antibody" as used herein is defined to include antibodies of
classes IgG, IgM, IgA, IgD, and IgE, and preferentially binding fragments
and hybrid derivatives of antibodies, including Fab and F(ab').sub.2
fragments of antibodies. Antibodies may be polyclonal or monoclonal.
Generally, monoclonal antibodies are preferred for use in the assays of
this invention.
Immunological methods are most convenient for carrying out the assays of
this invention because of their specificity, and the term "immunoassays"
as used herein is defined to mean any method using a preferential binding
of an antigen with a second material (i.e., a binding partner, usually an
antibody or antibody fragment having an antigen binding site) which binds
preferentially with an epitope of the antigen. Preferential binding as
used herein refers to binding between binding partners which is selective
and generally specific, and which demonstrates generally less than 10%,
preferably less than 5%, cross-reactive nonspecific binding. For example,
when the analyte is fetal fibronectin, the anti-(fetal fibronectin)
antibody is less than 10%, and preferably less than 5%, cross-reactive
with adult fibronectins.
Included within the scope of this invention are all immunoassays including
this step, including but not limited to sandwich, competition, dip stick,
agglomeration, precipitation, transistor bridge probe, particle sorting,
light disturbing, light scattering, and ultrasonic probe immunoassays, for
example. Appropriate immunoassays may use radioisotopes, enzymes, or
fluorogenic, chromogenic, or chemiluminescent substances, for example, as
labels.
A test sample which is to be assayed is removed in the vicinity of
posterior fornix, the cervical canal or cervical os, and the sample is
assayed to determine the presence or quantity of fetal restricted antigen
in the sample. Preferably, the presence of the antigen is determined. The
sample generally comprises fluid and particulate solids, and may contain
vaginal or cervical mucus, other vaginal or cervical secretions, cells or
cell debris, amniotic fluid, or other fetal or maternal materials. The
sample is removed with a swab having a dacron or other fibrous tip,
aspirator, suction device, lavage device or the like and is transferred to
a suitable container for storage and transport to the testing laboratory.
It is important that the test sample be dispersed in a liquid which
preserves the sensitive protein analytes which are unstable in the sampled
composition. The storage and transfer medium should prevent decline in the
protein analyte level during storage and transport. A suitable preserving
solution for storage and transfer consists of 0.05M Tris-HCl, pH 7.4;
0.15M NaCl, 0.02% NaN.sub.3, 1% BSA, 500 Kallikrein Units/mL of aprotinin,
1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA, and is described
in U.S. Pat. No. 4,919,889, issued Apr. 24, 1990. The solution is the most
preferred sample diluent solution when detecting fetal fibronectin.
Detection of fetal restricted antigen can be achieved by binding the fetal
restricted antigen in a test sample with an antibody which binds
preferentially with an epitope of the fetal restricted antigen, and
determining the presence or absence of this binding.
In one sandwich assay for fetal restricted antigen, the test sample is
contacted with an insoluble support to which anti-(fetal restricted
antigen) antibody is adhered to effect binding of fetal restricted antigen
in the sample to the insoluble support. The insoluble support is then
contacted with a secondary antibody, an unlabeled or labeled anti-(fetal
restricted antigen) antibody, which binds with the fetal restricted
antigen adhering to the insoluble support to detect and measure the
captured fetal restricted antigen.
An antibody which binds with a class of substances including the analyte
fetal restricted antigen can be substituted for a specific anti-(fetal
restricted antigen) antibody capture antibody or the specific anti-(fetal
restricted antigen) antibody sandwiching antibody. For example,
anti-(fetal fibronectin) antibody can be adhered to the insoluble support,
and labeled or unlabeled anti-(fibronectin) antibody can be used to detect
the captured antigen. Alternatively, anti-(fibronectin) antibody can be
adhered to the insoluble support, and labeled or unlabeled anti-(fetal
fibronectin) antibody is used to label the captured antigen. Preferably,
the anti-(fetal restricted antigen) antibody is used as the capture
antibody to ensure detection when the fetal restricted antigen is present
in small amounts in the sample in comparison to other antigens of the
class.
The secondary antibody can have a physically detectable label which can be
measured directly on the insoluble support. Alternatively, the secondary
antibody can be unlabeled, and the secondary antibody can be determined by
contacting the insoluble support with a labeled antibody or antibody
fragment which binds selectively with the secondary antibody (i.e., a
tertiary antibody), removing unbound labeled tertiary antibody from the
support, and determining the presence of the label on the insoluble
support. Sandwich immunoassays using a membrane substrate are appropriate
for use.
The sample can also be tested by a competition immunoassay procedure. The
test sample is mixed with labeled reagent antibody or antigen and
incubated with an insoluble support to which an anti-(fetal restricted
antigen) antibody or reagent fetal restricted antigen is adhered,
competition occurring between the reagents for binding with the sample
analyte. Methods and procedures to accomplish such immunoassays are well
known to those skilled in the immunoassay art. The label ultimately
adhering to the insoluble support or remaining in the solution is
determined.
Anti-(fetal restricted antigen) antibody can be obtained from fetal
restricted antigens, preferably from highly purified fetal restricted
antigens, by conventional antiserum or monoclonal techniques. This
invention will be described herein with respect to the detection of fetal
fibronectin as a fetal restricted antigen, for purposes of clarity, and
not by way of limitation: the detection of any fetal restricted antigen is
intended to be within the scope of this invention. Fetal fibronectin is
purified from amniotic fluid as described by Engvall and Ruoslahti, Int.
J. Cancer 20:1-5 (1977). Anti-(fetal fibronectin) antibody can be derived
from fetal fibronectin by conventional antiserum techniques or by
monoclonal antibody techniques.
Both monoclonal and polyclonal anti-(fetal restricted antigen) antibodies,
or anti-(fetal restricted antigen class) antibodies can be derived
directly from fetal restricted antigens, preferably from highly purified
antigens, by conventional antiserum or monoclonal techniques.
The principal antibodies useful in the assays of this invention are IgG and
IgM antibodies, although the IgD, IgE and IgA antibodies can also be used
if available in sufficient quantity. The antibodies are then affinity
purified using conventional affinity chromatography techniques such as
those described by Mishell and Shiigi in SELECTED METHODS IN CELLULAR
IMMUNOLOGY. San Francisco: Freeman (1980), Goding, J., MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE. New York: Academic Press pp 111-114
(1983), and Parikh et al, C&EN (Aug. 26, 1985).
Preferentially binding antibody fragments suitable for use in the kit and
method of this invention can be made from the respective monoclonal or
polyclonal antibodies by conventional enzyme or chemical fragmentation
procedures. Suitable procedures are described by Tijssen, P. LABORATORY
TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: PRACTICE AND THEORIES OF
ENZYME IMMUNOASSAYS. New York: Elsevier (1985), for example.
Polyclonal anti-(fetal restricted antigen) antibody can be obtained by
immunizing an animal such as a rabbit, guinea pig, rat or goat with
concentrated fetal restricted antigen, such as fetal fibronectin, removing
serum from the immunized animal, and separating the immunoglobulins from
the serum, for example by ammonium sulfate precipitation.
Suitable absorbents for use in affinity chromatography include cross-linked
agarose and cross-linked polyacrylamides to which the fetal restricted
antigen antibody is covalently bonded. For removal of antibodies
cross-reacting with adult fibronectins, the antibody serum is passed
through columns to which are coupled adult fibronectins. A portion of the
eluant containing the remaining antibody can then be passed through a
fetal fibronectin column and eluted to yield the affinity purified
antibody.
In these procedures, the antibody solution can be applied to the column in
a phosphate buffered saline solution, and the antibodies can be eluted
with a 2.5M NaSCN solution, pH 8.0. Antibody concentration, if desired,
can be achieved by negative pressure dialysis or ultrafiltration. The
antibody solution is stable at temperature of 4.degree. C. or less.
Repetition of the column separation procedures is continued until the
desired separation and purity is achieved.
For production of fetal fibronectin antigen and antibodies the procedures
described by H. Matsuura and S. Hakomori, Proc. Natl. Acad. Sci. USA
82:6517-6521 (1985) can be followed, replacing the tumor fibronectin with
fetal fibronectin. A most preferred anti-(fetal fibronectin) antibody is
produced by the hybridoma deposited at the American Type Culture
Collection and given the accession number ATCC HB 9018 is described in
detail in U.S. Pat. No. 4,894,326 issued Jan. 16, 1990 to Matsuura et al.
That monoclonal antibody is designated FDC-6. Culture of the hybridoma and
preparation of the antibodies for use in an immunoassay is described in
detail in the Examples.
Anti-(fetal restricted antigen class) antibodies of both polyclonal and
monoclonal varieties are generally well known and available either
commercially or from publicly available hybridoma deposits. For example,
anti-(fibronectin) monoclonal antibodies can be derived from clone samples
from ATCC HB 91 (American Type Culture Collection, Rockville, Md.). Other
such antibodies are described in Japanese Patent Application 60091264
(DIALOG database file 351, WPI Acc. No. 85-161617/27) and U.S. Pat. No.
4,325,867. A preferred procedure for preparation of polyclonal
anti-fibronectin antibodies in goats and rabbits is described in the
Examples.
Sandwich Immunoassays: In a sandwich embodiment of this invention to
determine fetal restricted antigen in a test sample, an insoluble support
to which anti-(fetal restricted antigen) antibody is adhered is contacted
with a test sample diluted with an aqueous buffer solution for a
sufficient time to permit binding of fetal restricted antigen in the test
sample with the anti-(fetal restricted antigen) antibody on the insoluble
support, and then removing the sample from the support. Suitable
immunoassay solutions are well known and include buffer solutions such as
phosphate buffer solution (PBS), pH 6 to 8 and preferably from 7.2 to 7.6.
Preferably, the sample is diluted in the sample diluent solution described
hereinbefore. The incubation time should be sufficient to permit
substantial binding to occur, the time being temperature dependent.
Suitable incubation times are from 30 to 240 minutes at temperatures
within the range of from 16.degree. to 40.degree. C., the preferred
contact time being at least 60 minutes at temperatures within the range of
from 20.degree. to 26.degree. C.
Residual sample solution is then removed from the support by use of a rinse
solution. Any conventional rinse solution can be used. A suitable rinse
solution is described in U.S. Pat. No. 4,528,267. It is an aqueous
phosphate buffer solution having a phosphate molarity of from 0.0001 to
0.05, a pH of from 6 to 8 and containing from 0.001 to 0.1 weight percent
of nonionic surfactant. Suitable nonionic surfactants include
polyoxyethylene ethers (BRIJ such as lauryl, cetyl, oleyl, stearyl, and
tridecyl polyoxyethylene ethers; polyoxyethylene sorbitans (TWEEN such as
polyoxyethylene sorbital monolaurate, monopalmitate, monostearate,
monoleate, and trioleates); and other polyoxyethylene ethers (TRITON, for
example). Preferred nonionic surfactants are octylphenoxypolyethoxy
ethanol having 40 ethylene oxide units (TRITON X-405, Rohm and Hass
Company) and polyoxyethylene sorbital monolaurate (TWEEN 20, available
commercially from Sigma Chemical Company). A most preferred rinse solution
is 0.02M Tris, 0.08M sodium chloride, 0.05% Tween-20, and 0.02% sodium
azide.
The insoluble support is then contacted with an antibody which will bind
with the captured fetal restricted antigen on the insoluble support, i.e.,
the sandwiching antibody. The sandwiching antibody can be an anti-(fetal
restricted antigen) antibody, or can be an anti-(fetal restricted antigen
class) antibody. The sandwiching antibody can be labeled or unlabeled. In
the event that an unlabele sandwiching antibody is used, a tertiary
antibody which binds with the sandwiching antibody and which bears a
physically detectable label can be used in a conventional manner to
determine the sandwiching antibody.
A variety of labels are described herein. For purposes of clarity and not
by way of limitation, the subsequent steps of the process will be
described for anti-(fetal restricted antigen) antibodies which have been
labeled with an enzyme, preferably a chromogenic or a fluorogenic enzyme.
The term "chromogenic enzyme" is defined herein to refer to an enzyme
which will produce a chromophore product with a suitable substrate. The
term "fluorogenic enzyme" is defined herein to refer to an enzyme which
will produce a fluorophore product with a suitable substrate.
The sandwiching antibody is applied to the insoluble support in an aqueous
solution. The solution preferably contains suitable salts and buffers to
preserve the reactants and facilitate the binding reaction. For example,
the solution can contain bovine serum albumin (BSA), phosphate buffer
solution (PBS), and a mild surfactant such as polyoxyethylene sorbitan
ester employed in the above-described rinse solution. A preferred diluent
for an enzyme-conjugated antibody is 0.05M Tris Buffer pH 7.2, 2%
D-Sorbitol, 2% BSA, 0.1% Sodium Azide, 0.01% Tween-20, 1 mM Magnesium
Chloride, and 0.1% Zinc Chloride.
The incubation is continued for sufficient time to permit the sandwiching
antibody to bind with exposed fetal restricted antigen epitopes, if any,
adhering to the insoluble support. The preferred incubation times and
temperatures are as set forth above for the binding of insolubilized
reagent anti-(fetal restricted antigen) antibody with the test sample
fetal restricted antigen.
The sandwiching antibody solution is then removed from the insoluble
support, and the support is rinsed with a rinse solution such as described
above, to remove any residual, unbound material.
If the sandwiching antibody is unlabeled, an enzyme labeled antibody or
other binding agent which binds selectively with the sandwiching antibody
is applied to the insoluble support in an aqueous solution. The solution
preferably contains suitable salts and buffers to preserve the reactants
and facilitate the binding reaction, such as described above. The
incubation is continued for sufficient time to permit labeled
anti-(sandwiching antibody) antibody to bind with exposed sandwiching
antibody epitopes, if any, adhering to the insoluble support. The
preferred incubation times and temperatures are as set forth for the
binding of insolubilized reagent anti-(fetal restricted antigen) antibody
with the test sample fetal restricted antigen.
The labeled antibody solution is then removed from the insoluble support,
and the support is rinsed with a rinse solution such as described above,
to remove any residual, unbound labeled material.
In a next step, the insoluble support is contacted with an aqueous solution
of a substrate which undergoes a reaction in the presence of the enzyme to
release fluorescent or chromogen compound into the solution. Suitable
substrates and the enzymes with which they can be converted are described
in U.S. Pat. Nos. 4,190,496 and 4,528,267, for example. The support is
contacted with an aqueous solution of the substrate containing from
10.sup.-2 to 10.sup.-10 molar concentrations of the substrate. Substrate
molar concentrations of from 10.sup.-4 to 10.sup.-5 are preferred.
Preferred additional reagents and buffers in the substrate solution
include 2-amino-2-methyl-1-propanol buffer and magnesium chloride, for
example.
The substrate solution is incubated with the insoluble support for
sufficient time for the reaction yielding the fluorophore or chromophore
to occur. At temperatures of from 18.degree. to 40.degree. C., incubation
times of from 5 to 240 minutes can be used. Preferably, the temperature is
within the range of from 20.degree. to 26.degree. C., and the incubation
time is from 10 to 90 minutes.
The fluorescent or chromophore level in the solution is then measured. The
equipment and procedures for determining the level of fluorescence or
chromophore level in the substrate solutions are those conventionally used
in the art. The level of fluorescence or chromogen in solution is a
function of the enzyme concentration on the insoluble support which is, in
turn, a function of the amount of fetal restricted antigen in the test
sample. The concentration of the fetal restricted antigen can be
determined by comparing the fluorescence or chromophore level of the
solution with respective fluorescence or chromophore levels obtained with
control solutions containing known concentrations of fetal restricted
antigen. Preferably, a control having a concentration of the fetal
restricted antigen which is statistically significantly different from
background is used. For fetal fibronectin, the control can be amniotic
fluid having a known fetal fibronectin concentration. The amniotic fluid
can be purified prior to use, if desired. The fetal fibronectin
concentration can vary from about 1 to about 1,000 ng/mL, preferably from
about 10 to 100 ng/mL, most preferably about 50 ng/mL. Samples having an
absorbance greater than or equal to that of the control are considered
positive. A preferred sandwich assay is described in detail in the
Examples.
The sandwich procedure can be modified to use a fetal restricted antigen
class binding antibody as either the capture or, preferably, the
sandwiching antibody. In these embodiments, an anti-(fetal restricted
antigen class) antibody, such as an anti-(fibronectin) antibody, is
adhered to the insoluble support, and a labeled or unlabeled anti-(fetal
restricted antigen) antibody is applied as the sandwiching antibody.
Preferably, the anti-(fetal restricted antigen) antibody can be adhered to
the insoluble support, and a labeled or unlabeled anti-(fetal restricted
antigen class) antibody is used to sandwich the captured antigen.
Membrane Immunoassays: In a membrane embodiment of this invention to
determine fetal restricted antigen in a test sample, an insoluble support
to which anti-(fetal restricted antigen) antibody is adhered is contacted
with a test sample diluted with an aqueous buffer solution such as
phosphate buffer solution (PBS), pH 6 to 8 and preferably from 7.2 to 7.6,
for a sufficient time to permit binding of fetal restricted antigen in the
sample with the anti-(fetal restricted antigen) antibody on the insoluble
support. The time required for binding is very small in a flow through
system. Suitable incubation times can be one second up to 20 minutes at
temperatures within the range of from 16.degree. to 40.degree. C., the
preferred contact time being less than one minute and optimally from 10
seconds to 2 minutes.
The insoluble support is then contacted with an antibody which will bind
with the captured fetal restricted antigen on the insoluble support, i.e.,
the sandwiching antibody. The sandwiching antibody can be labeled or
unlabeled. In the event that an unlabeled sandwiching antibody is used, a
tertiary antibody which binds with the sandwiching antibody and which
bears a physically detectable label can be used in a conventional manner
to determine the sandwiching antibody.
A variety of labels are described herein. For purposes of clarity and not
by way of limitation, the subsequent steps of the process will be
described for anti-(fetal restricted antigen) antibodies which have been
labeled with an enzyme, preferably a fluorogenic or chromogenic enzyme.
The sandwiching antibody is applied to the insoluble support in an aqueous
solution. The solution preferably contains suitable salts and buffers to
preserve the reactants and facilitate the binding reaction. For example,
the solution can contain bovine serum albumin (BSA), phosphate buffer
solution (PBS), and a mild surfactant such as polyoxyethylene sorbitan
ester employed in the above-described rinse solution. The incubation is
continued for sufficient time to permit the sandwiching antibody to bind
with exposed fetal restricted antigen epitopes, if any, adhering to the
insoluble support. The preferred incubation times and temperatures are as
set forth for the binding of insolubilized reagent anti-(fetal restricted
antigen) antibody with the test sample fetal restricted antigen.
The sandwiching antibody solution optionally can be removed from the
insoluble support, and the support is rinsed with a rinse solution such as
described above, to remove any residual, unbound labeled material.
If the sandwiching antibody is unlabeled, an enzyme labeled antibody or
other binding agent which binds selectively with the sandwiching antibody
is applied to the insoluble support in an aqueous solution. The solution
preferably contains suitable salts and buffers to preserve the reactants
and facilitate the binding reaction. For example, the solution can contain
bovine serum albumin (BSA), phosphate buffer solution (PBS), and a mild
surfactant such as polyoxyethylene sorbitan ester employed in the
above-described rinse solution. The incubation is continued for sufficient
time to permit labeled anti-(sandwiching antibody) antibody to bind with
sandwiching antibody epitopes, if any, adhering to the insoluble support.
The preferred incubation times and temperatures are as set forth for the
binding of insolubilized reagent anti-(fetal restricted antigen) antibody
with the test sample fetal restricted antigen.
The labeled antibody solution is then removed from the insoluble support,
and the support is rinsed with a rinse solution such as described above,
to remove any residual, unbound labeled material.
In a next step of the membrane sandwich process of this invention, the
insoluble support is contacted with an aqueous solution of a substrate
which undergoes a reaction in the presence of the enzyme to release
fluorogen or chromogen compound into the solution. Suitable substrates and
the enzymes with which they can be converted, as well as additional
components and buffers have been described above.
The substrate solution is incubated with the insoluble support for
sufficient time for the reaction yielding the fluorophore or chromophore
to occur. At temperatures of from 18.degree. to 40.degree. C., incubation
times of from 1 to 20 minutes can be used. Preferably, the temperature is
within the range of from 20.degree. to 26.degree. C., and the incubation
time is from 2 to 5 minutes. The fluorogen or chromogen level on the
membrane can be measured using a reflectometer or densitometer.
In an alternate membrane embodiment, an anti-(fetal restricted antigen)
antibody is bound to the membrane. The sample and a labeled anti-(fetal
restricted antigen class) antibody are combined. Following a time
sufficient for antibody binding, the sample/conjugate solution is
contacted with the membrane. Fetal restricted antigen in the sample binds
to the anti-(fetal restricted antigen) antibody, producing a sandwich of
anti-(fetal restricted antigen) antibody/fetal restricted antigen/labeled
anti-(fetal restricted antigen class) antibody on the membrane. In a
preferred embodiment, the label is colloidal gold.
Competition Immunoassays: Competition embodiments of this invention using
labeled reagent fetal restricted antigen comprise contacting a mixture of
the test sample and the labeled reagent fetal restricted antigen with an
anti-(fetal restricted antigen) antibody adhered to an insoluble support,
and determining the amount of label which either binds with the insoluble
support or remains in the solution phase.
The competition embodiments of this invention using labeled anti-(fetal
restricted antigen) antibodies can be of more than one form. One
embodiment using anti-(fetal restricted antigen) antibody bound to the
insoluble support comprises contacting a mixture of the test sample and
labeled anti-(fetal restricted antigen) antibodies with an anti-(fetal
restricted antigen) antibody adhered to an insoluble support, and
determining the amount of label which either binds with the insoluble
support or remains in the solution phase. Another embodiment using reagent
fetal restricted antigen bound to the insoluble support comprises
contacting a mixture of the test sample and labeled anti-(fetal restricted
antigen) antibodies with a fetal restricted antigen adhered to an
insoluble support, and determining the amount of label which either binds
with the insoluble support or remains in the solution phase.
In each of these methods, the test sample is diluted with buffer solution,
incubated and the label determined as described above with respect to the
sandwich immunoassay embodiments. The concentration of the limiting
reagent is selected to permit competitive binding between the reagents,
with the amount of label remaining on the insoluble support or in the
solution being a variable which is a function of the amount of the analyte
in the test sample. These methods are generally well known and how to vary
them to optimize a procedure are fully within the knowledge of a person
skilled in the immunoassay art.
The binding of the anti-(fetal restricted antigen) antibody and the fetal
restricted antigen in the test sample can also be determined by
agglomeration of particles to which the anti-(fetal restricted antigen)
antibody is adhered by fetal restricted antigen in the sample,
precipitation of antibodies due to antibody-antigen reactions, or
observations of physical or electrical changes which occur upon the
antibody-antigen binding, using semiconductor bridge probes, light
disturbing patterns such as are described in U.S. Pat. No. 4,647,544, and
the like.
Fetal Restricted Antigen Test Kits for determining fetal restricted antigen
in a test sample included within the scope of this invention generally
include an anti-(fetal restricted antigen) antibody adhered to an
insoluble support and an anti-(fetal restricted antigen class) antibody.
In a preferred embodiment the anti-(fetal restricted antigen) antibody is
a monoclonal antibody and the anti-(fetal restricted antigen class)
antibody is a polyclonal antibody. More preferably, the anti-(fetal
restricted antigen class) antibody is labeled with an enzyme, preferably
alkaline phosphatase. The kit can additionally contain enzyme substrate, a
positive control, a negative control, rinse buffer, or one or more sample
preparation devices such as sample filtering devices.
The kits of this invention can further comprise combinations to determine a
fetal restricted antigen in a test sample; buffers for sample transport,
storage and dilution; vials, foil packages or other containers of reagents
of this invention; other, optional reagents such as enzyme substrate
reagents in separate vials or other packages; mechanical or optical
devices to determine the presence and extent of antibody-antigen binding;
and combinations thereof. Sampling devices such as sampling swabs, and
buffers for transport and storage can also be included or packaged
separately. The individual parts of the kit may be packaged in any
convenient form, such as vials, foil packages, or other containers. For
example, insoluble support structures in a foil package can be combined
with other reagents in vials or other packages. A most preferred container
for the liquid reagents in the kit is a polyethylene dropper bottle
container which accurately dispenses an appropriate size drop; e.g. 50 or
100 .mu.L. A preferred kit is described in detail in the Examples.
FETAL RESTRICTED ANTIGEN PREGNANCY TEST
A pregnancy testing embodiment involves the detection of a fetal restricted
antigen, that is, a uniquely fetal or placental focused material, in a
test sample removed in the vicinity of the cervical canal or cervical os.
We have discovered that detectable amounts of these materials are present
in such a sample during the first 20 weeks of pregnancy. Since the fetal
restricted antigens are not present in significant quantities in the
maternal blood, the presence of maternal blood in the sample does not
interfere with the test.
The assay is performed as described above. A determination of the presence
of fetal restricted antigen in the test sample indicates pregnancy.
ECTOPIC PREGNANCY TEST
A method of this invention which determines ectopic pregnancy involves the
detection of the presence or absence of a fetal restricted antigen in a
test sample removed in the vicinity of the cervical canal and/or cervical
os. Detectable amounts of these materials are normally present in such
samples during the first 20 weeks of normal pregnancy. The absence of
fetal restricted antigen in such a sample from a pregnant woman during the
first 20 weeks of pregnancy is indicative of the presence of an ectopic
pregnancy. Since the fetal restricted antigens are not present in
significant quantities in the maternal blood, the presence of maternal
blood in the sample does not interfere with the test.
A test sample which is to be assayed is removed in the vicinity of the
cervical canal and/or cervical os, and the sample is assayed to determine
the presence or quantity of fetal restricted antigen in the sample as
described. It may also be desirable to determine pregnancy using the
presence of pregnancy-indicating hormones in serum or urine. A wide
variety of methods are known to a person skilled in the art for
determining pregnancy using the blood or urine of a patient. Any reliable
method can be used. Procedures for measuring hCG in plasma, serum and/or
urine are described in U.S. Pat. Nos. 3,171,783, 3,234,096, 3,236,732,
3,298,787, 3,309,275, 3,485,751, 3,655,838, 3,689,633, 3,862,302,
3,873,682, 3,873,683, 3,833,304, 3,991,175, 4,003,988, 4,014,653,
4,016,250, 4,033,723, 4,071,314, 4,094,963, 4,123,224, 4,123,509;
4,138,214, 4,208,187, 4,210,723, 4,234,561, 4,256,629, 4,268,435,
4,270,923, 4,310,455, 4,313,871, 4,320,111, 4,348,207, 4,371,515,
4,419,453, 4,421,896, 4,493,793, 4,508,829, and 4,665,034, for example.
Pregnancy detection by measuring progesterone metabolites in urine (U.S.
Pat. No. 3,141,740) or in milk, serum or plasma (Hungary Patent No.
T37028, WPI No. 86-023344/04); human placental lactogen in serum or plasma
(U.S. Pat. Nos. 3,892,841, 4,371,515, and 4,493,793 ); estrogen steroids
in urine (U.S. Pat. No. 3,955,928); luteinizing hormone (LH), prolactin
(PRL) and/or hCG-like substances in serum, plasma or urine (U.S. Pat. Nos.
4,016,250, 4,094,963, and 4,320,111); pregnancy specific .beta..sub.1
-glycoprotein (U.S. Pat. Nos. 4,065,445 and 4,191,533); LH (U.S. Pat. Nos.
4,138,214 and 4,208,187); bovine pregnancy antigen in bovine serum or
urine (European Patent Application 188,551, WPI No. 86-042108/06); a new
placental protein (U.S. Pat. No. 4,592,863); and early pregnancy factor WO
8605498 (WPI No. 86-264940/40) have been described. Methods have been
described for determining pregnancy by adding dyes to urine (U.S. Pat.
Nos. 2,587,221 and 3,226,196, dinitrophenylhydrazine; U.S. Pat. No.
3,595,620, bromocresol purple or chlorophenol red), by an iodine-paper
test (U.S. Pat. No. 3,248,173), by adding other precipitating agents (U.S.
Pat. No. 3,278,270), by a treatment of female blood with a mixture of
acids and sodium chloride (U.S. Pat. No. 3,883,304). Pregnancy may be
determined using an assay of a test sample removed in the vicinity of the
cervical canal or cervical os following the teachings of U.S. application
Ser. No. 121,902 filed Nov. 17, 1987. Any one of the above methods can be
used, but methods such as hCG measurements are preferred.
A determination of the presence of pregnancy together with a negative
determination of fetal restricted antigen in the test sample during the
first 20 weeks of pregnancy indicates the absence of normal uterine
pregnancy, and is an indication that an ectopic pregnancy has occurred.
EX VIVO PRODUCTS OF CONCEPTION TEST
An embodiment of this invention which determines the presence of ex vivo
products of conception involves the detection of a fetal restricted
antigen in a test sample expelled by a suspected spontaneous abortion or
removed during a dilation and curettage or a therapeutic abortion
procedure. Since the fetal restricted antigens are not present in
significant quantities in maternal blood, the presence of maternal blood
in the sample does not interfere with the tests.
A test sample which is to be assayed for the presence of ex vivo products
of conception is procured which is believed to represent material expulsed
from the uterus. Such materials can be tissues removed during a
therapeutic abortion or a D&C (dilation and curettage). Alternatively, the
material may be vaginal discharge which is believed to be indicative of a
spontaneous abortion, or miscarriage. The sample generally comprises fluid
and particulate solids, and may contain tissue matter, vaginal or cervical
mucus, other vaginal or cervical secretions, cells or cell debris,
amniotic fluid, or other fetal or maternal materials. The sample can be
procured from the vaginal cavity with a swab having a dacron or other
fibrous tip, aspirator, suction device, lavage device or the like. The
sample can represent tissues removed during a dilation and curettage
procedure or a therapeutic abortion, or can be procured using a feminine
protection pad in the case of a suspected spontaneous abortion. It is
important that the test sample be dispersed in a liquid which preserves
the sensitive protein analytes as described hereinbefore.
Detection of fetal restricted antigen can be achieved by the methods
described hereinbefore. It may be desirable to additionally determine
pregnancy using the presence of pregnancy-indicating hormones in serum or
urine. Any reliable method can be used, such as those described above with
reference to the Ectopic Pregnancy Test.
A test sample obtained during a D&C, therapeutic abortion, or suspected
spontaneous abortion (miscarriage) can be tested for the presence of fetal
restricted antigen. Concurrent determinations of the presence of
pregnancy, and negative determination of fetal restricted antigen in a
test sample suspected of representing a therapeutic or spontaneous
abortion, is an indication that pregnancy has occurred, and is continuing.
Determination of the presence of pregnancy, and positive determination of
fetal restricted antigen in a test sample suspected of representing a
therapeutic or spontaneous abortion is an indication that pregnancy has
occurred, but has been terminated. Determination of the absence of both of
pregnancy indicators, and negative determination of fetal restricted
antigen in a test sample derived from a D&C is an indication that
pregnancy has not occurred, and that no pregnancy has been terminated.
RISK OF PRETERM LABOR/RUPTURED MEMBRANE TEST
An embodiment of this invention for indicating increased risk of preterm
birth involves the detection of a fetal restricted antigen in a test
sample after week 20 of pregnancy. We have discovered that detectable
amounts of these materials are generally absent from vaginal samples such
as those obtained from the vicinity of the posterior fornix, cervical
canal or cervical os after week 20 of pregnancy. Detectable amounts of
these materials present in such samples taken after week 20 pregnancy
indicate an increased risk of impending preterm birth and/or indicate the
rupture of the amniotic membrane. Since the fetal restricted antigens are
not present in significant quantities in the maternal blood, the presence
of maternal blood in the sample does not interfere with the test.
A test sample which is to be assayed is removed from the vicinity of the
posterior fornix, cervical canal or cervical os, and the sample is assayed
to determine the presence or quantity of fetal restricted antigen in the
sample as described hereinbefore. A determination of the presence of fetal
restricted antigen in a test sample after 20 weeks of pregnancy indicates
that fetal membranes may have ruptured, and/or is an indication of
increased risk of preterm labor.
INSOLUBLE SUPPORTS
Antigen and antibody reagents of this invention can be bonded to insoluble
supports by conventional processes. Antigen binding methods suitable for
binding antigens to insoluble supports such as those described in U.S.
Pat. Nos. 3,234,096, 3,236,732, 3,309,275, 3,873,683, 3,991,175,
4,003,988, 4,016,250, 4,033,723, 4,071,314, 4,348,207, and 4,419,453, for
binding antigens to latex particles and erythrocytes, for example, can be
used. Procedures for binding of antibodies to insoluble supports are
described in U.S. Pat. Nos. 3,551,555, 3,553,310, 4,048,298 and Re.
29,474, and by Tijsson, PRACTICE AND THEORY OF ENZYME IMMUNOASSAYS.
Elsevier Science Publishers, (1985) pp 297-328, for example. Procedures
for binding of antibodies to polystyrene by adsorption are described in
U.S. Pat. Nos. 3,646,346 and 4,092,408, for example. For purposes of
clarity and not by way of limitation, the binding procedures are described
herein with respect to the binding of antibodies to insoluble supports.
These procedures are suitable for binding of reagent antibodies, such as
anti-(fetal restricted antigen) antibodies and anti-(fetal restricted
antigen class) antibodies, and for binding of reagent antigens such as
reagent fetal restricted antigen to insoluble supports.
A variety of materials can be used as the insoluble support, the primary
consideration being the binding of the antibody to the surface, and the
absence of interference with the antigen binding reaction or with other
reactions which can be employed to determine the presence and extent of
the binding reaction. Organic and inorganic polymers, both natural and
synthetic, can be used as the insoluble support. Examples of suitable
polymers include polyethylene, polypropylene, polybutylene,
poly(4-methylbutylene), butyl rubber, silastic polymers, polyesters,
polyamides, cellulose and cellulose derivatives (such as cellulose
acetate, nitrocellulose and the like), acrylates, methacrylates, vinyl
polymers (such as polyvinyl acetate, polyvinyl chloride, polyvinylidene
chloride, polyvinyl fluoride, and the like), polystyrene and styrene graft
copolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, etc. Other
materials which can be used as the insoluble support can the latexes of
the above polymers, silica gel, silicon wafers, glass, paper, insoluble
protein, metals, metalloids, metal oxides, magnetic materials,
semi-conductive materials, cermets and the like. In addition are included
substances which form gels, such as proteins such as gelatins,
lipopolysaccharides, silicates, agarose, polyacrylamides or polymers which
form several aqueous phases such as dextrans, polyalkylene glycols
(alkylene with 2 to 3 carbon atoms) or surfactants, e.g. amphophilic
compounds such as phospholipids, long chain (12-24 carbon atoms) alkyl
ammonium salts and the like.
A preferred insoluble support of this invention comprises a nylon or
nitrocellulose membrane. Alternate insoluble supports are made from a
polystyrene, styrene copolymers such as styrene-acrylonitrile copolymers,
or polyolefins such as polyethylene or polypropylene, and acrylate and
methacrylate polymers and copolymers. The most preferred insoluble
supports of this invention are nylon membranes or polystyrene microtiter
plates. The reagent antibody or antigen reagents can be bound to the
insoluble support by adsorption, ionic bonding, van der Waals adsorption,
electrostatic bonding, or other non-covalent bonding, or it can be bound
to the insoluble support by covalent bonding.
A particularly advantageous support for this procedure comprises a
microtiter plate having a plurality of wells. The well surface or plastic
cup inserts therein can constitute the antigen or antibody support. If the
determination will require the use of fluorometric measurements, the
microtiter plate or the well inserts are advantageously opaque to light so
that excitation light applied to a well does not reach or influence
contents of the surrounding wells.
Procedures for non-covalent bonding are described in U.S. Pat. No.
4,528,267. Procedures for covalently bonding antibodies and antigens to
insoluble supports are described by I. Chibata in IMMOBILIZED ENZYMES.
Halsted Press: New York (1978) and A. Cuatrecasas, J. Bio. Chem. 245:3059
(1970). The surface can be coated with a protein and coupled with the
antibody or antigen using procedures described in U.S. Pat. No. 4,210,418
using glutaraldehyde as a coupling agent, for example. In an alternate
procedure, the well can be coated with a layer having free isocyanate
groups such as a polyether isocyanate, and application of the antibody or
antigen in aqueous solution thereto effects the requisite bonding. In yet
another procedure, the antibody or antigen can be coupled to a
hydroxylated material by means of cyanogen bromide as described in U.S.
Pat. No. 3,720,760.
The insoluble supports are preferably "blocked" to reduce nonspecific
binding. The choice of suitable blocking agents is determined by the type
of insoluble support. For example, for polystyrene supports, suitable
blocking agents include water-soluble non-immune animal proteins. Suitable
water-soluble non-immune animal proteins include bovine serum albumin
(BSA); human, rabbit, goat, sheep, and horse serum albumins; casein and
non-fat milk; ovalbumin; glycoproteins; and the like. A most preferred
blocking/stabilizing solution is 4% sucrose, 1% mannitol, 0.5% casein,
0.01M PBS.
The same blocking agents can also be used for nylon and nitrocellulose
supports. However, a preferred blocking agent for nitrocellulose or nylon
membrane supports is non-fat milk or casein. An optimum blocking agent for
these membrane supports is an aqueous solution containing from 1 to 5 wt.
% non-fat dried milk or casein, and nonionic surfactants such as
polyoxyethylene sorbitan derivatives and polyoxyethylene ethers.
LABELED REAGENTS
The labeled reagent fetal restricted antigen, anti-(fetal restricted
antigen) antibody, anti-(fetal restricted antigen class) antibody and
anti-(sandwiching antibody) reagent antibodies of this invention can be
prepared by conventional procedures for attaching labels to proteins,
preferably with suitable protection of antibody binding sites.
The labels can be bonded or coupled to the protein reagents by chemical or
physical bonding. Ligands and groups which can be conjugated to the
reagent antigen or antibodies of this invention include elements,
compounds or biological materials which have physical or chemical
characteristics which can be used to distinguish the reagents to which
they are bonded from compounds and materials in the sample being tested.
Labeling procedures are described herein with respect to labeling
antibodies for purposes of clarity and not by way of limitation, and the
procedures described are generally suitable for labeling any proteinaceous
compound or substance, such as the reagent pregnancy antigens or fetal
restricted antigens herein.
Radiolabeled antibodies of this invention can be used for in vitro
diagnostic tests. The specific activity of a tagged antibody depends upon
the half-life, isotopic purity of the radioactive label and how the label
is incorporated into the antigen or antibody. Table A lists several
commonly used isotopes, their specific activities and half-lives. In
immunoassay tests, the higher the specific activity, in general, the
better the sensitivity.
TABLE A
______________________________________
Specific Activity of Pure
Isotope Isotope (Curies/mole)
Half-Life
______________________________________
.sup.14 C
6.25 .times. 10.sup.1
5720 years
.sup.3 H
2.91 .times. 10.sup.4
12.5 years
.sup.35 S
1.50 .times. 10.sup.6
87 days
.sup.125 I
2.18 .times. 10.sup.6
60 days
.sup.32 P
3.16 .times. 10.sup.6
14.3 days
.sup.131 I
1.62 .times. 10.sup.7
8.1 days
______________________________________
Procedures for labeling antibodies with radioactive isotopes listed in
Table A are generally known in the art. Tritium labeling procedures are
described in U.S. Pat. No. 4,302,438, for example. Iodinating, tritium
labeling and .sup.35 S labeling procedures especially adapted for
antibodies are described by Goding, J., MONOCLONAL ANTIBODIES: PRINCIPLES
AND PRACTICE New York: Academic Press (1983) pp 124-126, and the
references cited therein. Other procedures for iodinating antibodies are
described by Hunter and Greenwood, Nature 144:945 (1962) and David et al,
Biochem. 13:1014-1021 (1974) and in U.S. Pat. Nos. 3,867,517 and
4,376,110. Examples of suitable systems, coupling procedures and substrate
reactions therewith are disclosed in U.S. Pat. Nos. Re. 31,006, 3,654,090,
4,214,048, 4,289,747, 4,302,438, 4,312,943, 4,376,110 and the references
cited therein, for example. Examples of other suitable systems are
described by Pesce et al, Clin. Chem. 20:353-359 (1974) and Wisdom, G.,
Clin. Chem. 22:1243 (1976).
A list of suitable enzyme classes which can be used for labeling, and
specific examples for each class, follow:
TABLE B
______________________________________
Class Enzyme Example
______________________________________
Hydrolases Amylases
Nucleases Polynucleotidase
Amidases Arginase
Purine deaminases Adenase
Peptidases Aminopolypeptidase
Proteinases Pepsin
Esterases Lipases
Iron Enzymes Catalase
Copper Enzymes Tyrosinases
Enzymes containing Coenzymes
Alcohol dehydrogenase
Enzymes reducing cytochrome
Succinic dehydrogenase
Yellow enzymes Diaphorase
Mutases Glyoxalase
Desmolases Aldolase
Oxidases Glucose oxidase
Horseradish peroxidase
Phosphatases Alkaline Phosphatases
Acid Phosphatases
Dehydrogenases G6PDH (Glucose 6
phosphodehydrogenase)
.beta.-galactosidase
Phosphorylases
Hexokinases
______________________________________
Suitable enzymes are described in Hawk et al, PRACTICAL PHYSIOLOGICAL
CHEMISTRY, New York: McGraw-Hill pp. 306-397 (1954).
Fluorogenic and chromogenic enzymes (enzymes in the presence of which a
selected substrate will produce a fluorescent or chromogenic product) are
useful labeling moieties. Methods for selectively conjugating enzymes to
antibodies without impairing the ability of the antibody to bind with
antigen and for conjugating enzymes to proteinaceous reagents are well
known in the art.
Suitable enzymes and procedures for coupling them to antibodies are
described by I. Chibata in IMMOBILIZED ENZYMES. Halsted Press: New York
(1978); A. Cuatrecasas, J. Bio. Chem. 245:3059 (1970); Wilson, M. et al,
INTERNATIONAL CONFERENCE IN IMMUNOFLUORESCENCE AND RELATED STAINING
TECHNIQUES. W. Knapp et al, editors. Amsterdam: Elsevier pp. 215-244
(1978); Sullivan, M. et al, Ann. Clin. Biochem. 16:221-240 (1979); Nygren,
H. et al, Med. Biol. 57:187-191 (1979); Gadkari, D. et al, J. Virol. Meth.
10:215-224 (1985); Tijssen, P. et al, Anal. Biochem. 136:451-457 (1984);
Tsuruta, J. et al, J. Histochem. Cytochem. 33:767-777 (1985); Ishikawa,
E., J. Immunoassay 4:209-327 (1983); and in U.S. Pat. No. 4,190,496, for
example.
The preferred enzymes and suitable substrates corresponding thereto include
horseradish peroxidase for which suitable substrates are
o-phenylenediamine, m-phenylenediamine, o-dianisidine, and
4-chloro-.alpha.-napthol. They also include .beta.-galactosidase for which
suitable substrates are 4-methylumbelliferyl-.beta.-D-galactoside,
p-nitrophenyl-.beta.-D-galactose, p-nitrophenol,
o-nitrophenyl-.beta.-D-galactose, and o-nitrophenol, for example. They
include alkaline phosphatase for which suitable substrates are
p-nitrophenylphosphate, indoxyl phosphate, and 5-bromo-3-chloroindoxyl
phosphate, for example. A most preferred enzyme substrate combination is
alkaline phosphatase and phenolphthalein monophosphate.
Examples of suitable procedures for enzyme labeling the antibody include
the use of carbodiimides, daldehydes, and bifunctional coupling reagents.
Linkage of enzymes through amide groups can be achieved by treating the
proteins with thionyl chloride, N-hydroxysuccinimide or similar reagents
in an anhydrous solvent such as dimethylformamide, dioxane,
dimethylsulfoxide, tetrahydrofuran, or the like. Alternative coupling
agents include carbodiimides such as
1-ethyl-3-(3-(N,N'-dimethylamino)propyl)carbodiimide,
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate,
succinimidyl 4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, and
succinimidyl 3-(2-pyridyldithio)-propionate, for example.
The carbohydrate moiety of an enzyme can also be oxidized to an aldehyde
and reacted with lysyl amino groups of immunoglobulins to form a Schiffs
base. Reduction with sodium borohydride effects a stable linkage of enzyme
and antibody. Horseradish peroxidase with antibody can be efficiently
linked to immunoglobulins by the method of Wilson, M. et al, INTERNATIONAL
CONFERENCE IN IMMUNOFLUORESCENCE AND RELATED STAINING TECHNIQUES. W. Knapp
et al, editors. Amsterdam: Elsevier pp 215-244 (1978).
Fluorophore and chromophore labeled antibodies can be prepared from
standard fluorescent moieties known in the art. Since antibodies and other
proteins absorb light having wavelengths up to about 310 nm, the
fluorescent moieties should be selected to have substantial absorption at
wavelengths above 310 nm and preferably above 400 nm. A variety of
suitable fluorescers and chromophores are described by Stryer, Science
162:526 (1968) and Brand, L. et al, Ann. Rev. Biochem. 41:843-868 (1972).
The antibodies can be labeled with fluorescent chromophore groups by
conventional procedures such as those disclosed in U.S. Pat. Nos.
3,940,475, 4,289,747 and 4,376,110, for example.
One group of fluorescers having a number of the desirable properties
described above are the xanthene dyes, which include the fluoresceins
derived from 3,6-dihydroxy-9-phenylxanthhydrol and resamines and
rhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanime
rhodamine B. The rhodamine and fluorescein derivatives of
9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluorescein
compounds having reactive coupling groups such as amino and isothiocyanate
groups such as fluorescein isothiocyanate and fluorescamine are readily
available.
Another group of fluorescent compounds are the naphthylamines, having an
amino group in the alpha or beta position. Included among the
naphthylamino compounds are 1-dimethylaminonaphthyl-5-sulfonate,
1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene
sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin; acridines
such as 9-isothiocyanatoacridine and acridine orange;
N-[p-(2-benzoxazolyl)phenyl]maleimide; benzoxadiozoles such as
4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and
7-(p-methoxybenzylamino)-4-nitrobenzo-2-oxa-1,3-diazole; stilbenes such as
4-dimethylamino-4'-isothiocyanatostilbene and
4-dimethylamino-4'-maleimidostilbene;
N,N'-dioctadecycloxacarboxyamine-p-toluenesulfonate; pyrenes such as
8-hydroxy-1,3,6-pyrenetrisulfonic acid, 1-pyrenebutyric acid, merocyanine
540, rose bengal, 2,4-diphenyl-3(2H)-furanone, o-phthaldehyde, as well as
other readily available fluorescing molecules. These dyes either have
active functionalities or such functionalities can be readily introduced.
Antibodies can be labeled with fluorochromes or chromophores by the
procedures described by Goding, J., MONOCLONAL ANTIBODIES: PRINCIPLES AND
PRACTICE. New York: Academic Press (1983) pp 208-249. The concentration of
fluorochrome is selected according to the table of Goding, supra, p 229.
For example, fluorescein isocyanate (1.0 mg/mL) or rhodamine isocyanate
(10.0 mg/mL) in DMSO is prepared, and the desired volume (1-10% of total
protein solution volume) is added to the protein solution dropwise, with
stirring. The reaction proceeds for two hours, shielded from light. The
product is purified by gel filtration on SEPHADEX G-25 gel in PBS
containing 0.1% NaNO.sub.3 to separate the unreacted or hydrolyzed
fluorochrome. The absorbance of the conjugate is measured at 280 nm and at
its peak in the visible region (495 nm for fluoresceinated antibody and
550 nm for rhodaminated antibody). The fluorochrome to protein ratio is
calculated according to the procedure of Goding, MONOCLONAL ANTIBODIES:
PRINCIPLES AND PRACTICE. New York Academic Press (1983) pp 224-225.
Conjugates are stored at 4.degree. C. protected from light until use. If
the antibody solution concentration is less than 1 mg/mL, BSA is added to
the solution to a final concentration of 1 mg/mL.
The antibodies and reagent antigens used in the assays of this invention
can be covalently bonded to avidin or biotin. Suitable binding procedures
involve cross-linking through a bifunctional cross-linking agent. Suitable
bifunctional compounds are described by Peters, K. et al, Ann. Rev.
Biochem. 46:523 (1977). Alkyl imidates show a high degree of specificity
among the functional groups presented to them by a protein. The reaction
is specific for primary amino groups. Examples of suitable coupling
reagents include amidoesters such as dimethylmalonimidate, azides such as
the acyl azide of tartryl diazide which reacts readily with immuno groups
to produce amide linkages. Aryl dihalides (e.g.,
1,5-difluoro-2,4-dinitrobenzene, or 4,4'-difluoro- 3,3'-dinitrophenyl
sulfone, glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, dimaleimide, mixed anhydride, m-maleamidobenzoyl
N-hydroxysucciinimide ester, and other known cross-linking agents can be
used.
The foregoing reagents provide essentially irreversible bonds. Bifunctional
agents with functional groups such as disulfide or glycol may be used.
These provide bonds which can be broken after the cross-linking reaction,
if desired. Such reagents include dimethyl 3,3'-dithiobispropionimidate,
succinimidylpropionimidate, N-(3-fluoro-4,6-dinitrophenyl)cystamine,
tartryl diazide, tartryl di(glycylazide) and tartryl di(epsilon-amino
caproylazide).
In other instances, the bonds can be formed directly between the reagents
themselves. For example, antibody can be bound to biotin through
functional groups on the respective materials. As a specific example,
biotin can be treated with periodate and reacted with antibody to give a
Schiff base formation without inhibiting the biotin to avidin binding or
blocking immunological activity of the antibody. Avidin-conjugated and
biotinylated reagents are available from Vector Laboratories, Burlingame,
Calif.
Known techniques using bifunctional cross-linking agents include the
following: (a) a one-step glutaraldehyde linkage, Avrameas, S.,
Immunochem. 6:43 (1969); (b) two-step glutaraldehyde linkage, Avrameas,
S., Immunochem. 8:1175 (1971); and (c) dimaleimide linkage, Kato, K. et
al, Euro. J. Biochem. 62:285 (1966).
Antibodies can be labeled with metallic radionuclides according the
procedure of Hnatowich, D. et al. J. Appl. Rad. 35:554-557 (1984) and
Buckley, R et al. Fed. Eur. Biochem. Soc. 166:202-204 (Jan. 1984). In this
procedure the antibodies are conjugated with a chelating agent, such as
diethylenetriaminepentaacetic acid (DTPA), which is capable of forming a
chelate with the metallic radionuclide. A suspension of 0.1 mg/mL of the
bicyclic anhydride of DTPA is prepared in a dry solvent such as
chloroform, ether or dry DMSO. An aliquot is removed to a clean, dry tube
sufficient to provide a DTPA to immunoglobulin molar ratio of 1:1 and
evaporated under nitrogen. A 10-20 microliter portion of the antibody
solution used (10-20 mg/mL) in 0.05M bicarbonate buffer in saline, pH
7.0-7.5, is added to the dry DTPA, and the contents are agitated for
0.5-1.0 minute. The coupled protein preparation is diluted to 0.2 mL with
the same buffer solution and purified on a 5 cm gel filtration column with
SEPHADEX G-50 gel, using a saline eluant. The coupling efficiency is
determined before purification by the addition of "chelation-grade"
.sup.111 In in 0.5M acetate buffer solution, pH 6.0. Thin layer
chromatography is used to separate the DTPA coupled antibody for
calculation of the coupling efficiency. The DTPA-coupled antibodies can be
stored at 4.degree. C. until needed for binding with metallic
radionuclides such as .sup.111 IN+3, .sup.212 Bi+3 and .sup.68 Ga+3, for
example.
This invention is further illustrated by the following specific, but
non-limiting examples. Temperatures are given in degrees Centigrade and
percents as weight percents unless otherwise specified.
EXAMPLE 1
Polyclonal Anti-(fetal fibronectin) Antibody
Fetal fibronectin is purified from amniotic fluid as described by Engall
and Ruoslahti, Int. J. Cancer 20:1-5 (1977).
The anti-(fetal fibronectin) antibodies are elicited in rabbits using the
immunization techniques and schedules described in the literature, e.g.,
Stollar, Meth. Enzym. 70:70 (1980), immunizing the rabbits with the fetal
fibronectin antigen. The antiserum is screened in a solid phase assay
similar to that used for monoclonal antibodies, e.g., as described by
Lange et al, Clin. Exp. Immunol. 25:191 (1976) and Pisetsky et al, J.
Immun. Meth. 41:187 (1981).
The IgG fraction of the antisera is purified further by affinity
chromatography using CNBr-Sepharose 4B (Pharmacia Fine Chemicals) to which
has been coupled fetal fibronectin. The method used for coupling is that
recommended by the gel manufacturer, AFFINITY CHROMATOGRAPHY. Pharmacia
Fine Chemicals, pp 15-18.
The column is equilibrated with from 2 to 3 volumes of buffer (0.01M PBS,
pH 7.2), and the anti-(fetal fibronectin) antibody containing solution is
then applied to the column. The absorbency of the eluate is monitored at
280 nm until protein no longer passes from the column. The column is then
washed with 0.1M glycine buffer, pH 2.5, to desorb the immunoaffinity
bound anti-(fetal fibronectin) antibody. Peak protein fractions are
collected, pooled and dialyzed against 0.01M PBS, pH 7.2, for 24-36 hr at
4.degree. C. with multiple buffer changes.
If a higher purity is desired, the affinity purified IgG can be passed
through an adult plasma fibronectin bound affinity column by the procedure
described above to remove any antibodies which would cross-react with
adult plasma fibronectins.
EXAMPLE 2
Monoclonal Anti-(fetal fibronectin) Antibody
Using the purified fetal fibronectin obtained by the procedure of Example
1, mouse monoclonal antibodies to the fetal fibronectin are obtained using
standard procedures of Galfre and Milstein, Meth. Enzym. 73:1 (1981) and
Matsuura, H. and Hakomori, S. et al, Proc. Natl. Acad. Sci. USA
82:6517-6521 (1985), using fetal fibronectin as the antigen for immunizing
the mice. The monoclonal antibodies are screened using a modification of
the techniques described in the literature, e.g., Lange et al,
Clin.Exp.Immunol. 25:191(1976) and Pisetsky et al, J. Immun. Meth. 41:187
(1981).
Mouse monoclonal antibody is purified from ascites fluid or from hybridoma
culture supernatants using Protein-A coupled Sepharose-4B (Pharmacia Fine
Chemicals) according to the procedure of Tijsson, PRACTICE AND THEORY OF
ENZYME IMMUNOASSAYS. Elsevier Science Publishers, pp 105-107 (1985).
EXAMPLE 3
Polyclonal Anti-(fetal fibronectin) Antibody-Coated Microtiter Plate
Rabbit anti-(fetal fibronectin) prepared and further purified to remove
adult fibronectin cross-reactivity as described in Example 1 is diluted to
10 .mu.g/mL in 0.05M carbonate buffer, pH 9.6. 100 .mu.L is dispersed into
each well of an IMMULON II microtiter plate (Dynatech). The plate is
covered and incubated 4 hr at room temperature or 4.degree. C. overnight.
The plate is washed 4 times with Wash Buffer (0.02M Tris HCl, 0.015M NaCl,
0.05% TWEEN-20), filling and emptying the wells completely with each use.
The plate is then blocked by dispersing into each well 200 .mu.L of a
blocking solution (0.01M PBS, 1% BSA, 0.02% NaN.sub.3, pH 7.4) and
incubating for 1 hr at room temperature. The wells are then washed 4 times
with Wash Buffer, as described above. The plate is now ready for
immunoassay of samples.
EXAMPLE 4
Polyclonal Anti-Human Fibronectin Antibody
Human plasma fibronectin was purified from human plasma as described by
Engvall and Ruoslahti, Int. J. Cancer 20:1-5 (1977).
The anti-human plasma fibronectin antibodies were elicited in goats using
the immunization techniques and schedules described in the literature,
e.g., Stollar, Meth. Enzym. 70:70 (1980), immunizing the goats with the
human plasma fibronectin antigen. The antiserum was screened in a solid
phase assay similar to that used for monoclonal antibodies, e.g., as
described by Lange et al, CIin. Exp. Immunol. 25:191 (1976) and Pisetsky
et al, J. Immun. Meth. 41:187 (1981).
The IgG fraction of the antiserum was purified further by affinity
chromatography using CNBr-Sepharose 4B (Pharmacia Fine Chemicals) to which
has been coupled human plasma fibronectin according to the method
recommended by the manufacturer (AFFINITY CHROMATOGRAPHY, Pharmacia Fine
Chemicals Catalogue 1990), pp 15-18.
Briefly, the column was equilibrated with from 2 to 3 volumes of buffer
(0.01M PBS, pH 7.2), and the anti-human fibronectin antibody-containing
solution was then applied to the column. The absorbency of the effluent
was monitored at 280 nm until protein no longer passed from the column.
The column was then washed with equilibration buffer until a baseline
absorbance at 280 nm was obtained.
The immunoaffinity bound anti-human plasma fibronectin antibody was eluted
with 0.1M glycine buffer, pH 2.5. Peak protein fractions were collected,
pooled and dialyzed against 0.01M PBS, pH 7.2, for 24-36 hr at 4.degree.
C. with multiple buffer changes.
The above procedure was repeated to immunize rabbits with human plasma
fibronectin and to purify the resultant polyclonal anti-human fibronectin
antibodies.
EXAMPLE 5
Polyclonal Anti-Fibronectin
Antibody-Coated Microtiter Plate
Goat anti-human plasma fibronectin prepared as described in Example 4 is
diluted to 10 .mu.g/mL in 0.05M carbonate buffer, pH 9.6. 100 .mu.L is
dispersed into each well of a polystyrene microtiter plate such as
supplied by Costar, Nunc, or Dynatech. The plate is covered and incubated
2 to 4 hr at room temperature or 4.degree. C. overnight. The plate is
washed 3 to 4 times with Wash Buffer (0.02M Tris HCl, 0.015M NaCl, 0.05%
TWEEN-20), filling and emptying the wells completely with each use. The
plate is then blocked by dispersing into each well 200 .mu.L of a
blocking/stabilizing solution (4% sucrose, 1% mannitol, 0.01M PBS, 1% BSA,
0.02% NaN.sub.3, pH 7.4) and incubated for 30 minutes to 2 hrs at room
temperature. The wells are then aspirated to dryness, the plate is
packaged in an air-tight container with a desiccant pouch, and stored at
4.degree. C. until needed.
EXAMPLE 6
Monoclonal Antibodies from Hybridoma HB 9018
Preparation of the Hybridoma deposited at the American Type Culture
Collection and given the accession number ATCC HB 9018 is described in
detail in U.S. Pat. No. 4,894,326 issued Jan. 16, 1990 to Matsuura et al,
which patent is incorporated herein by reference in its entirety.
The hybridoma was cultured by growth in RPMI 1640 tissue culture medium
supplemented with 10% fetal bovine serum. Additionally, the hybridoma was
cultured in mice by the injection of the hybrid cells according to the
procedure of Mishell and Shiigi (Selected Methods in Cellular Immunology,
W.H. Freeman & Co, San Francisco p 368, 1980).
The monoclonal antibody designated FDC-6 and produced by the hybridoma was
prepared for use in an immunoassay by the following procedure. The IgG
fraction of the culture supernatant or the ascites was precipitated by
ammonium sulfate fractionation. The antibody was redissolved and dialyzed
into the appropriate buffer for purification by affinity chromatography on
Protein-G Fast Flow (Pharmacia Fine Chemicals) according to the
manufacturer's directions.
EXAMPLE 7
Monoclonal Antibody-Coated Microtiter Plate
Microtiter plates were coated with FDC-6 monoclonal antibody by following
the procedure described below.
Monoclonal antibody FDC-6 prepared as described in Example 6 was diluted to
10 .mu.g/ml in phosphate buffer, pH 7.2 and 100 .mu.l/well was dispersed
into a polystyrene microtiter plate (Costar). The plates were incubated
for 2 hours at room temperature or overnight at 4.degree. C. The contents
of the wells were aspirated and the wells washed 3 to 4 times with wash
buffer (0.02M Tris HCl, 0.015M NaCl, 0.05% TWEEN-20) as described in
Example 5.
200 .mu.l/well of blocking/stabilizing solution (4% sucrose, 1% mannitol,
0.5% casein, 0.01M PBS) was then added to the wells and incubated for 30
minutes to 4 hours at room temperature. The wells were then aspirated to
dryness, and the plate was packaged in an air-tight container with a
desiccant pouch, and stored at 4.degree. C. until needed.
The above procedure was repeated using microtiter plates from Nunc and
Dynatech and gave equivalent results.
EXAMPLE 8
Enzyme Labeled Anti-(fibronectin) Antibody
Anti-human plasma fibronectin antibody prepared according to Example 4 was
conjugated with alkaline phosphatase following the one-step glutaraldehyde
procedure of Avrameas, Immunochem. 6:43 (1969).
EXAMPLE 9
Fetal Fibronectin Assay Kit and Method
In a preferred embodiment, an assay kit for the fetal restricted antigen,
fetal fibronectin included the following reagents:
1. a microtiter plate coated with murine monoclonal anti-fetal fibronectin
antibody.
2. alkaline phosphatase-conjugated, affinity purified, polyclonal, goat
anti-fibronectin antibodies
3. enzyme substrate
4. a negative control
5. a positive control
6. rinse buffer concentrate (50X)
The microtiter plate coated with murine monoclonal anti-fetal fibronectin
antibody and the alkaline phosphatase-conjugated, affinity purified,
polyclonal, goat anti-fibronectin antibodies were prepared as described in
Examples 7 and 8, respectively. The microtiter plate was packaged as 12
strips of eight wells each in sealed plastic bags containing desiccant.
The stock antibody conjugate was appropriately diluted in conjugate diluent
(0.05M Tris Buffer pH 7.2, 2% D-Sorbitol, 2% BSA, 0.1% Sodium Azide, 0.01%
Tween-20, 1 mM Magnesium Chloride, and 0.1% Zinc Chloride) and 10 ml
placed in a polyethylene dropper bottle container.
The enzyme substrate (10 mL in a polyethylene dropper bottle container) was
phenolphthalein monophosphate (1 mg/ml) dissolved in 0.4M
aminomethylpropanediol buffer, pH 10 with 0.1 mM magnesium chloride and
0.2% sodium azide.
The positive control (2.5 mL in a polyethylene dropper bottle container)
was amniotic fluid containing fetal fibronectin diluted to a concentration
of fetal fibronectin of 50 ng/mL in sample diluent solution (0.05M Tris
buffer pH 7.4, 1% bovine serum albumin (BSA), 0.15M sodium chloride, 0.02%
Sodium Azide, 5 mM ethylenediamine tetraacetic acid (EDTA), 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 500 Kallikrein Units/ml of
Aprotinin). This sample diluent solution is described in U.S. Pat. No.
4,919,889 to Jones et al, issued Apr. 24, 1990, which patent is
incorporated herein by reference in its entirety.
The negative control (2.5 mL in a polyethylene dropper bottle container)
was the sample diluent solution used for the positive control without
fetal fibronectin.
The rinse buffer (10 mL in a polyethylene dropper bottle container) was a
50X concentrate containing 1.0M Tris buffer pH 7.4, 4.0M sodium chloride,
2.5% Tween-20, and 1% sodium azide. The rinse buffer was diluted with
water to a final concentration of 0.02M Tris, 0.08M sodium chloride, 0.05%
Tween-20, and 0.02% sodium azide for use in the assay.
The kit additionally contained 24 5.mu. pore size polyethylene sample
filters (Porex Technologies, Fairburn, Ga.), a microtiter strip holder, a
microtiter plate cover and an instruction sheet. All of the dropper
bottles in the kit were polyethylene bottles designed to dispense
approximately 50 .mu.L drops of the reagent. All of the assay steps
performed following sample collection utilized the reagents and materials
in the kit.
The assay was performed as follows. All samples were collected in the
vicinity of the posterior fornix or cervical os using dacron swabs. Swab
samples were immersed in 1.0 mL of sample diluent in a collection vial.
The sample diluent solution is described above. The swabs were removed
from the solution leaving as such liquid as possible in the collection
tube. The samples were incubated at 37.degree. C. along with the controls
from the assay kit for 15 minutes prior to the assay, either before or
after filtration. A sample filter was snapped in place on each sample
tube. The 8-well strips were snapped into place in a strip holder. The
holder had the alphanumeric indications of the 12 columns and eight rows
of standard microtiter plates. Duplicate 100 .mu.L aliquots of each sample
and the positive and negative controls were placed in separate wells of
the microtiter strip and incubated for 1 hour at room temperature.
Following incubation, samples and controls were aspirated from the wells.
Wells were washed three times with diluted wash buffer (1X). Following
washing, 100 .mu.L of enzyme-antibody conjugate was added to each well and
incubated for 30 minutes at room temperature. The wells were aspirated and
washed as described above. Following washing, 100 .mu.L of enzyme
substrate was added to each well and incubated for 30 minutes at room
temperature.
Following the incubation, the plates were gently agitated by hand or with
an orbital shaker to mix the well contents. The frame of strips was placed
in an ELISA plate reader. The absorbance of each well at 550 nm was
determined. The average absorbance of the duplicate wells for each sample
and control was calculated. If the absorbance of the patient sample was
less than the absorbance of the positive control, the sample was negative,
indicating an undetectable level of fetal fibronectin in the sample. If
the sample absorbance is greater than or equal to the absorbance of the
positive control, the sample was positive, indicating that fetal
fibronectin was present in the sample. In any assay if the absorbance of
the positive control was not greater than 1.5 times the absorbance of the
negative control the results were discarded and the assay procedure was
repeated.
EXAMPLE 10
Pregnancy Test
To perform the pregnancy test, a sample is removed from the vaginal cavity
in the vicinity of the cervical canal or cervical os, and assayed to
determine the presence of the fetal restricted antigen, fetal fibronectin
from a woman who is suspected of being pregnant. Samples obtained before
week 20 of pregnancy which demonstrate significant fetal fibronectin in
the test sample indicate normal uterine pregnancy.
Swab samples were obtained from 393 women as described in Example 9. Of the
women tested 50 were confirmed as non-pregnant (NP) (by analysis of serum
or urine human chorionic gonadoropin [hCG]); 333 were confirmed to have
intra-uterine pregnancies (IUP) (by analysis of serum or urine hCG); and
10 were confirmed to have ectopic pregnancies (ECT) (by medical history,
serum hCG, clinical examination and surgical confirmation).
The assay was performed as described in Example 9 with the following
exceptions. The antibody conjugate was goat anti-human fibronectin
(Jackson ImmunoResearch Labs catalogue no. 109-056-059) diluted 1:1,000 in
0.02M Tris, 0.3 m NaCl, 0.05% Tween 20, 5.0% BSA, 0.02% NaN. The enzyme
substrate was para-nitrophenyl phosphate (Sigma Chemical Co. catalogue no.
104-40T) diluted in AMP buffer (Sigma Chemical Co. catalogue no. 221). In
addition, the samples were centrifuged rather than filtered to remove
particulates. For this test, an assay which detected any fetal fibronectin
in the sample was scored as a positive test. The results of the tests are
illustrated below.
##STR1##
Analyses of the test results are illustrated below. The first analysis does
not include the results from women with ectopic pregnancies. The second
analysis includes results from women with ectopic pregnancies. In the
analyses, the following abbreviations were used. "Se" means sensitivity
(the number of true positive test results divided by the total number of
women with the condition; i.e., the number of true positive test results
divided by the the sum of the number of true positive and false negative
test results). "Sp" means specificity (the number of true negative test
results divided by the total number of women without the condition; i.e.,
the number of true negative test results divided by the sum of the number
of true negative and false positive test results). "PPV" means positive
predictive value (the number of true positive test results divided by the
total number of samples which tested positive). "NPV" means negative
predictive value (the number of true negative test results divided by the
total number of samples which tested negative).
##STR2##
The results of the studies demonstrate that a positive assay result
indicates that a woman is pregnant.
EXAMPLE 11
Ectopic Pregnancy Test
The procedure of Example 10 was repeated using the same samples. However,
in this case, a cutoff of 0.5 .mu.g/ml of fetal fibronectin (the value of
a negative control plus two standard deviations) was used for a positive
test result for the presence of fetal fibronectin. The data was analyzed
as described in Example 10. Sensitivity, specificity, positive predictive
value and negative predictive value are based on detection of ectopic
pregnancy in this analysis.
##STR3##
The results demonstrate that a positive test result provides a high degree
of confidence that a woman does not have an ectopic pregnancy. This is,
for these samples 100% of the test results indicating that a woman had an
intra-uterine pregnancy were correct. The test can therefore be
characterized as a "Rule in IUP" test; Specifically, a fetal fibronectin
concentration of >0.5 .mu.g/ml indicates an intra-uterine pregnancy.
EXAMPLE 12
Product of Therapeutic Abortion Test
Samples obtained from a therapeutic abortion were tested to verify that
fetal materials had been removed from the uterus. The assay was performed
as in Example 9 with the following exceptions. The samples were prepared
as follows. Samples were collected from products of conception by aqueous
filtration through woven cotton into a test tube, centrifuged at 2000 rpm
for 10 minutes and the supernatants assayed directly for fetal fibronectin
with no further dilution. A calibration curve was included in the test.
The calibrators were diluted from amniotic fluid of known fetal
fibronectin concentration to an assay range of from 10 ng/mL to 4 mg/mL.
Sample diluent solution was used as the negative background control. The
samples were assayed as described in Example 9 using the calibration curve
to quantify the fetal fibronectin concentration. A fibronectin cutoff of
0.11 .mu.g/ml (negative control value plus 2 standard deviations) was used
to determine positivity.
In this study, D&C materials from 291 women with confirmed intra-uterine
pregnancies and 8 women without uterine pregnancies (2 ectopically
pregnant and 6 nonpregnant women were evaluated). The results are
illustrated below.
##STR4##
In samples containing products of conception, significant amounts of fetal
fibronectin were found, confirming the existence of normal pregnancy and
the termination thereof. The data also demonstrate that in a pregnant
patient having a negative assay result, the possibility of an ectopic
pregnancy is indicated.
EXAMPLE 13
Preterm Labor Sandwich Immunoassay
The procedure of Example 9 was repeated with test samples obtained during
weeks 20-36 of pregnancy. Studies were conducted at three perinatal
referral clinics in the United States. Women were evaluated for admission
to the hospital for either suspected preterm rupture of membranes or
suspected preterm labor with intact membranes.
Confirmation of rupture of membranes was made by visual examination of the
vagina for gross pooling of amniotic fluid, microscopic examination of
dried vaginal secretions for ferning, presence of alkaline vaginal
secretions using nitrazine paper and ultrasound diagnosis of
oligohydramnios. Rupture of membranes was defined by the presence of any
two of these four diagnostic criteria. One hundred-seventeen women with
intact amniotic membranes pregnant between 23 weeks and 36 weeks, 6 days
of gestation based on last known menstrual period and expected date of
confinement confirmed by first trimester pelvic examination and
ultrasonography <28 weeks gestation are subsequently described. Women were
determined by the attending physician to be at risk for preterm labor and
subsequent delivery based on medical history and clinical examination
including recording of uterine contractions and examination of the cervix.
Since the clinical definition of preterm labor is sometimes difficult to
establish, data establishing the clinical utility of fetal fibronectin
were analyzed using preterm delivery as the outcome variable.
To assess the potential for cervicovaginal contamination by maternal plasma
fibronectin, maternal blood specimens were obtained from 52 women with
apparently healthy pregnancies during second or third trimester. Amniotic
fluid specimens were obtained from 92 patients undergoing amniocentesis
for genetic diagnosis in early second trimester and 8 patients undergoing
amniocentesis for evaluation of fetal lung maturity prior to elective
repeat, cesarean section in third trimester.
The assay results indicated that the concentration of fetal fibronectin in
amniotic fluid in second trimester was 87.1.+-.4.8 .mu.g/ml (n=92) and
27.1.+-.17.3 .mu.g/ml (n=8) in third trimester. The concentration of fetal
fibronectin in maternal plasma in the second trimester was 1.48.+-.0.11
.mu.g/ml (n=20) and 3.19.+-.0.30 .mu.g/ml (n=32) in the third trimester.
##STR5##
As is shown in the table above for the 117 patients with suspected preterm
labor and intact amniotic membrnes, 49 of 59 (sensitivity=83.1%) women
delivering prematurely (PTD) had fetal fibronectin in their cervicovaginal
secretions compared to 11 of 58 women (specificity=81.0%) delivering at
term (TD) (p<0.01). Similarly, those patients with fetal fibronectin in
their cervicovaginal secretions were far more likely to deliver
prematurely (positive predictive value=81.7%) than those women not
expressing cervicovaginal fetal fibronectin (negative predictive
value=82.5%).
The presence of cervicovaginal fetal fibronectin was a sensitive and
specific predictor of the risk for preterm delivery in these women with
suspected preterm labor. The presence of fetal fibronectin in these
patients was strongly associated with risk of preterm delivery with a
logistic regression odds ratio of 3.79 (95% CI:2.33, 6.15; p<0.01).
To evaluate for potential confounding by fetal fibronectin of maternal
origin, the data was analyzed after exclusion of 31 samples contaminated
with blood. As shown below, similar proportions of patients had fetal
fibronectin in their cervicovaginal secretions and delivered prematurely.
Furthermore, inclusion of the presence or absence of vaginal bloody show
into the stepwise logistic regression model gave an odds ratio of 1.70
(95%CI: 0.91,3.18; p=0.1) demonstrating that bloody show was not an
independent predictor of preterm delivery after fetal fibronectin was
introduced into the model. It was clear, however, from univariate analysis
that detection of fetal fibronectin in cervicovaginal secretions
contaminated with blood is an indicator of imminent delivery.
##STR6##
The utility of fetal fibronectin for identifying women at risk for PTD was
maintained even when women in preterm contractions with intact membranes
with cervical dilation exceeding 2 cm were eliminated from the analysis.
The logistic regression odds ratio of 3.18 (95%CI: 1.8,5.6, p<0.01)
confirmed the predictive value of fetal fibronectin in this clinically
discrete population.
##STR7##
EXAMPLE 14
Ruptured Membranes Sandwich Immunossay
The procedure of Example 9 was repeated with test samples obtained from
week 20 of pregnancy to term. The purpose of this multisite clinical study
was to evaluate the efficacy of the immunoassay to detect fetal
fibronectin in vaginal secretions of women with term pregnancies and
suspected rupture of membranes (TROM), women with preterm pregnancies and
suspected rupture of membranes (PROM) and pregnant women in third
trimester with intact amniotic membranes (CONTROL). The tenative diagnosis
of rupture of amniotic membranes was made according to the clinical
criteria outlined in Example 13. The assay results were analyzed for each
group by demographic characteristics, obstetrical history and salient
features of the current pregnancy including interval between sample
collection and delivery. Fetal fibronectin was analyzed in cervicovaginal
secretions obtained from 85 women in PROM, 339 women in TROM and 67 women
in CONTROL. Only data for women with known gestational ages as confirmed
by ultrasonography or last known menstrual period who gave informed
consent are subsequently described.
The following table shows the number of observations and means (.+-.SD) for
gestational ages (weeks) at sampling (EGAS) and delivery (EGAD) and the
interval between sampling and delivery (SAMDEL) for women in PROM, TROM
and CONTROL partitioned by fetal fibronectin result. Data is also provided
for TROM and CONTROL describing the percentage of deliveries occuring
within 48 hours of sampling (%Del<48Hrs) and the percentage of preterm
deliveries in PROM (%PTD).
__________________________________________________________________________
PROM TROM CONTROL
fFN +
fFN -
fFN +
fFN -
fFN +
fFN -
__________________________________________________________________________
n 80 5 319 20 13 54
EGAS 32.3 30.4 39.3 38.9 38.6 38.4
(Weeks) (3.8)
(4.5)
(1.8)
(1.3)
(1.4)
(1.5)
EGAD 32.7 33.5 39.4 39.9 39.6 40.3
(Weeks) (4.0)
(6.0)
(1.8)
(1.4)
(1.8)
(1.5)
SAMDEL 59.0 542.0
18.1 163.4
169.3
333.4
(Hours) (204)
(439)
(48) (182)
(165)
(213)
% Del < 48 Hrs
-- -- 94.7 45.0 23.1 5.3
% PTD 97.5 60.0 -- -- -- --
__________________________________________________________________________
Of the 85 patients in PROM with suspected preterm rupture of membranes, 80
had fetal fibronectin in their cervicovaginal fluid and 97.5% (n=78)
delivered prematurely indicating that the amniotic membranes were
ruptured. Of the 339 patients in TROM with suspected term rupture of
membranes, 319 had fetal fibronectin in their cervicovaginal fluid and
94.7% (n=302) delivered within 48 hours of sampling indicating that the
amniotic membranes were ruptured.
Of the 67 patients in CONTROL with apparently intact amniotic membranes, 13
had fetal fibronectin in their cervicovaginal fluid and 23.1% delivered
within 48 hours of sampling compared to 5.3% for the women in CONTROL with
negative fetal fibronectin results. These results indicate that
conventionally used diagnostic tests for detection of rupture of membranes
are frequently unreliable. Moreover, all women with positive fetal
fibronectin results had significantly (p<0.05) shorter sample to delivery
intervals than women with negative fetal fibronectin results.
Of the 339 samples collected from women in TROM, information regarding the
presence of vaginal bloody show was available for 316. Of these, 90
(28.5%) were collected in the presence of vaginal bloody show. EGAS, EGAD,
SAMDEL and %Del<48 hrs are shown for these women in the following table.
While EGAS and EGAD are similar for women with and without vaginal bloody
show, women with vaginal bloody show deliver more quickly than those
without blood in the vagina (p<0.05). The proportion of positive results
is similar regardless of the presence or absence of blood in the vagina at
the time of specimen collection.
______________________________________
TROM
Blood +
Blood -
______________________________________
n 90 226
EGAS 39.3 39.3
(Weeks) (1.0) (1.1)
EGAD 39.4 39.4
(Weeks) (1.1) (1.2)
SAMDEL 12.8 28.1
(Hours) (23.7) (111)
% Del < 48 Hrs 91.2 95.6
______________________________________
This analysis demonstrates that the presence of blood in vaginal secretions
has no apparent effect on test outcome for this population of women. Only
one woman in CONTROL was identified as having vaginal bloody show. She had
a negative assay result and delivered approximately 135 hours following
specimen collection.
Fetal fibronectin is an optimal marker for detection of amniotic fluid
indicating rupture of amniotic membranes. Fetal fibronectin is present in
amniotic fluid in high concentration and in the maternal blood in low
concentration. Immunologic detection of fetal fibronectin in
cervicovaginal fluid is a safe and effective method for identifying the
presence or absence of amniotic fluid in the vagina to determine if the
amniotic membranes have been compromised.
EXAMPLE 15
Fetal Fibronectin Assay Kit and Method
In another preferred embodiment, an assay kit for the fetal restricted
antigen, fetal fibronectin included the following components. This kit was
designed to be used to perform a rapid, bedside assay.
1. an assay device comprising a plastic housing and containing: (a) a
porous nylon membrane to which is bound a monoclonal anti-fetal
fibronectin antibody; (b) a flow control membrane system; and (c) an
absorbent layer
2. a colloidal gold-labeled goat anti-fibronectin antibody conjugate in a
protein matrix
3. conjugate reconstitution buffer
4. a wash solution
5. a sterile, dacron sample collection swab
The membrane device was prepared by the following procedure. Approximately
2 .mu.L of the murine monoclonal antibody FDC-6 prepared as described in
Example 6 is applied to a membrane surface (1.2.mu. nylon, Biodyne-A,
Pall) in a pH 6, 0.01M phosphate buffered saline (PBS), 0.1M citrate
buffer containing 0.5 mg/ml BSA. A procedural control consisting of human
plasma fibronectin purified as described in Example 4 in the same buffer
is also applied to a discrete region of the membrane. After the membrane
has air dried, a blocking reagent of PBS-buffered, 0.5% nonfat dry milk is
added to the membrane. The excess blocking reagent is removed after at
least about 20 minutes.
The membrane-holding device (Target Device, V-Tech, Pomona, Calif.) is
assembled with a second porous layer (0.45.mu. low protein-binding nylon,
LoProdyne, Pall) beneath the antibody-bearing membrane (in the direction
of sample application) for controlling the flow of sample solution from
the assay membrane to the absorbent layer. The two porous membranes are
then placed over an absorbent porous polyethylene layer having a capacity
of greater than 1.5 ml (Chromex, Brooklyn, N.Y.) and enclosed in the
device. The device is packaged individually in a sealed plastic bag
containing desiccant.
The colloidal gold is prepared by the reduction of 0.01% tetrachloroauric
acid with 0.16% sodium citrate in a manner which produces approximately 30
nm particles. Briefly, the two solutions are heated separately to
90.degree. C. The reducing solution is added to the gold solution while
vigorously stirring. The combined solution is boiled (100.degree. C.) for
at least 10 minutes.
Affinity purified goat anti-fibronectin antibody (prepared as described in
Example 4) was bound to the colloidal gold by adsorption. Briefly, the
collodial gold solution prepared above was combined with the antibody
(5-10 .mu.g/mL) in water. Following conjugation, the conjugate solution
was stabilized by the addition of 5% BSA and 5% polyvinylpyrrolidine
(final concentration).
The stock conjugate was concentrated approximately 10- to 12-fold by
ultrafiltration using a hollow fiber filter. The concentrated conjugate
was diluted to an appropriate level in 15 mM Tris, 2% BSA, 0.1% Tween 20,
0.2% polyethylene glycol, 8% polyvinylpyrrolidine and 0.04% thimerosal. An
appropriate concentration was determined by using a range of dilutions in
a sample assay procedure as described below and determining the dilution
which produces the best result.
The selected conjugate dilution is placed in polyethylene sample collection
tubes and lyophilized. The tubes are fitted with 2.mu. pore size
polyethylene sample filters (Porex Technologies, Fairburn, Ga.) during the
lyophilization process. The lyophilized conjugate is individually packaged
in a foil pouch with desiccant.
The conjugate reconstitution buffer is 100 mM sodium acetate. This buffer
is packaged as a unit dose in a 1 ml disposable tube.
The wash solution is water packaged as a unit dose in a disposable tube.
The kit additionally contains an individually packaged sterile dacron swab
and a procedural summary card.
The assay was performed as follows.
1. Before collecting the sample, remove the plastic tube containing gold
conjugate from the foil pouch, remove the dropper tip and add the entire
contents of the tube containing the conjugate reconstitution buffer.
2. Collect the sample with the swab provided. During a sterile speculum
examination, insert the swab into the posterior fornix of the vagina,
twirl for approximately 10 seconds to absorb fluid. Immediately proceed to
perform the test. Samples may not be stored for later testing. Place the
swab in the gold conjugate solution and mix rapidly with an up and down
motion for 10 to 15 seconds.
3. Remove as much liquid as possible from the swab by rolling the tip on
the inside of the tube. Dispose of the swab in a manner consistent with
handling potentially infectious materials.
4. Replace the dropper tip on the plastic tube and immediately dispense the
entire volume of diluted filtered sample onto the surface of the membrane
device.
5. After the sample liquid has been absorbed into the membrane surface, add
a few drops of wash solution and observe the results.
6. A negative result is indicated by a red color in the procedural control
area of the membrane only. A positive result is indicated by a pink or red
spot in the test zone of the membrane as well as in the control zone.
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